Methods for the treatment of cancer and inflammatory diseases using cereblon as a predictor

ABSTRACT

Uses of the protein cereblon as a predictor of clinical sensitivity to cancer, inflammatory diseases, and patient response to drug treatment.

The present application is a continuation of U.S. patent applicationSer. No. 13/459,005, filed Apr. 27, 2012, which claims priority to U.S.Provisional Patent Application No. 61/481,066, filed Apr. 29, 2011;61/511,986, filed Jul. 26, 2011; and 61/579,600, filed Dec. 22, 2011;the entirety of each of which is incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 5, 2012, isnamed 12827227.txt, is 119,198 bytes in size, and serves as both thepaper copy required by 37 C.F.R. 1.821(c) and the CRF required by 37C.F.R. 1.821(e).

1. FIELD

Provided herein are uses of the protein cereblon as a predictor ofclinical sensitivity to cancer and inflammatory diseases, and patientresponse to drug treatment.

2. BACKGROUND 2.1 Pathobiology of Cancer

Cancer is characterized primarily by an increase in the number ofabnormal cells derived from a given normal tissue, invasion of adjacenttissues by these abnormal cells, or lymphatic or blood-borne spread ofmalignant cells to regional lymph nodes and to distant sites(metastasis). Clinical data and molecular biologic studies indicate thatcancer is a multistep process that begins with minor preneoplasticchanges, which may under certain conditions progress to neoplasia. Theneoplastic lesion may evolve clonally and develop an increasing capacityfor invasion, growth, metastasis, and heterogeneity, especially underconditions in which the neoplastic cells escape the host's immunesurveillance. Roitt, I., Brostoff, J and Kale, D., Immunology,17.1-17.12 (3rd ed., Mosby, St. Louis, Mo., 1993).

There is an enormous variety of cancers which are described in detail inthe medical literature. Examples include cancers of the lung, colon,rectum, prostate, breast, brain, blood and intestine. The incidence ofcancer continues to climb as the general population ages, as new cancersdevelop, and as susceptible populations (e.g., people infected with AIDSor excessively exposed to sunlight) grow. However, options for thetreatment of cancer are limited. For example, in the case of bloodcancers (e.g., multiple myeloma), few treatment options are available,especially when conventional chemotherapy fails and bone-marrowtransplantation is not an option. A tremendous demand therefore existsfor new methods and compositions that can be used to treat patients withcancer.

Many types of cancers are associated with new blood vessel formation, aprocess known as angiogenesis. Several of the mechanisms involved intumor-induced angiogenesis have been elucidated. The most direct ofthese mechanisms is the secretion by the tumor cells of cytokines withangiogenic properties. Examples of these cytokines include acidic andbasic fibroblastic growth factor (a,b-FGF), angiogenin, vascularendothelial growth factor (VEGF), and TNF-α. Alternatively, tumor cellscan release angiogenic peptides through the production of proteases andthe subsequent breakdown of the extracellular matrix where somecytokines are stored (e.g., b-FGF). Angiogenesis can also be inducedindirectly through the recruitment of inflammatory cells (particularlymacrophages) and their subsequent release of angiogenic cytokines (e.g.,TNF-α, b-FGF).

Lymphoma refers to cancers that originate in the lymphatic system.Lymphoma is characterized by malignant neoplasms of lymphocytes—Blymphocytes and T lymphocytes (i.e., B-cells and T-cells). Lymphomagenerally starts in lymph nodes or collections of lymphatic tissue inorgans including, but not limited to, the stomach or intestines.Lymphoma may involve the marrow and the blood in some cases. Lymphomamay spread from one site to other parts of the body. The treatment ofvarious forms of lymphomas are described, for example, in U.S. Pat. No.7,468,363, the entirety of which is incorporated herein by reference.Such lymphomas include, but are not limited to, Hodgkin's lymphoma,non-Hodgkin's lymphoma, cutaneous B-cell lymphoma, activated B-celllymphoma, diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma(MCL), follicular center lymphoma, transformed lymphoma, lymphocyticlymphoma of intermediate differentiation, intermediate lymphocyticlymphoma (ILL), diffuse poorly differentiated lymphocytic lymphoma(PDL), centrocytic lymphoma, diffuse small-cleaved cell lymphoma(DSCCL), peripheral T-cell lymphomas (PTCL), cutaneous T-Cell lymphomaand mantle zone lymphoma and low grade follicular lymphoma.

Non-Hodgkin's lymphoma (NHL) is the fifth most common cancer for bothmen and women in the United States, with an estimated 63,190 new casesand 18,660 deaths in 2007. Jemal A, et al., CA Cancer J Clin 2007;57(1):43-66. The probability of developing NHL increases with age andthe incidence of NHL in the elderly has been steadily increasing in thepast decade, causing concern with the aging trend of the US population.Id. Clarke C A, et al., Cancer 2002; 94(7):2015-2023.

Diffuse large B-cell lymphoma (DLBCL) accounts for approximatelyone-third of non-Hodgkin's lymphomas. While some DLBCL patients arecured with traditional chemotherapy, the remainder die from the disease.Anticancer drugs cause rapid and persistent depletion of lymphocytes,possibly by direct apoptosis induction in mature T and B cells. See K.Stahnke. et al., Blood 2001, 98:3066-3073. Absolute lymphocyte count(ALC) has been shown to be a prognostic factor in follicularnon-Hodgkin's lymphoma and recent results have suggested that ALC atdiagnosis is an important prognostic factor in diffuse large B-celllymphoma. The diffuse large-B-cell lymphomas (DLBCL) can be divided intodistinct molecular subtypes according to their gene profiling patterns:germinal-center B-cell-like DLBCL (GCB-DLBCL), activated B-cell-likeDLBCL (ABC-DLBCL), and primary mediastinal B-cell lymphoma (PMBL) orunclassified type. These subtypes are characterized by distinctdifferences in survival, chemo-responsiveness, and signaling pathwaydependence, particularly the NF-κB pathway. See D. Kim et al., Journalof Clinical Oncology, 2007 ASCO Annual Meeting Proceedings Part I. Vol25, No. 18S (June 20 Supplement), 2007: 8082. See Bea S, et al., Blood2005; 106: 3183-90; Ngo V. N. et al., Nature 2011; 470: 115-9. Suchdifferences have prompted the search for more effective andsubtype-specific treatment strategies in DLBCL.

Leukemia refers to malignant neoplasms of the blood-forming tissues.Various forms of leukemias are described, for example, in U.S. Pat. No.7,393,862 and U.S. provisional patent application No. 60/380,842, filedMay 17, 2002, the entireties of which are incorporated herein byreference. Although viruses reportedly cause several forms of leukemiain animals, causes of leukemia in humans are to a large extent unknown.The Merck Manual, 944-952 (17^(th) ed. 1999). Transformation tomalignancy typically occurs in a single cell through two or more stepswith subsequent proliferation and clonal expansion. In some leukemias,specific chromosomal translocations have been identified with consistentleukemic cell morphology and special clinical features (e.g.,translocations of 9 and 22 in chronic myelocytic leukemia, and of 15 and17 in acute promyelocytic leukemia). Acute leukemias are predominantlyundifferentiated cell populations and chronic leukemias more mature cellforms.

Acute leukemias are divided into lymphoblastic (ALL) andnon-lymphoblastic (ANLL) types. The Merck Manual, 946-949 (17^(th) ed.1999). They may be further subdivided by their morphologic andcytochemical appearance according to the French-American-British (FAB)classification or according to their type and degree of differentiation.The use of specific B- and T-cell and myeloid-antigen monoclonalantibodies are most helpful for classification. ALL is predominantly achildhood disease which is established by laboratory findings and bonemarrow examination. ANLL, also known as acute myelogenous leukemia oracute myeloid leukemia (AML), occurs at all ages and is the more commonacute leukemia among adults; it is the form usually associated withirradiation as a causative agent.

Chronic leukemias are described as being lymphocytic (CLL) or myelocytic(CML). The Merck Manual, 949-952 (17^(th) ed. 1999). CLL ischaracterized by the appearance of mature lymphocytes in blood, bonemarrow, and lymphoid organs. The hallmark of CLL is sustained, absolutelymphocytosis (>5,000/μL) and an increase of lymphocytes in the bonemarrow. Most CLL patients also have clonal expansion of lymphocytes withB-cell characteristics. CLL is a disease of middle or old age. In CML,the characteristic feature is the predominance of granulocytic cells ofall stages of differentiation in blood, bone marrow, liver, spleen, andother organs. In the symptomatic patient at diagnosis, the total whiteblood cell (WBC) count is usually about 200,000/μL, but may reach1,000,000/μL. CML is relatively easy to diagnose because of the presenceof the Philadelphia chromosome.

Bone marrow stromal cells are well known to support CLL diseaseprogression and resistance to chemotherapy. Disrupting the interactionsbetween CLL cells and stromal cells is an additional target of CLLchemotherapy.

In addition to the acute and chronic categorization, neoplasms are alsocategorized based upon the cells giving rise to such disorder intoprecursor or peripheral. See e.g., U.S. patent publication no.2008/0051379, the disclosure of which is incorporated herein byreference in its entirety. Precursor neoplasms include ALLs andlymphoblastic lymphomas and occur in lymphocytes before they havedifferentiated into either a T- or B-cell. Peripheral neoplasms arethose that occur in lymphocytes that have differentiated into either T-or B-cells. Such peripheral neoplasms include, but are not limited to,B-cell CLL, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma,mantle cell lymphoma, follicular lymphoma, extranodal marginal zoneB-cell lymphoma of mucosa-associated lymphoid tissue, nodal marginalzone lymphoma, splenic marginal zone lymphoma, hairy cell leukemia,plasmacytoma, diffuse large B-cell lymphoma and Burkitt lymphoma. Inover 95 percent of CLL cases, the clonal expansion is of a B celllineage. See Cancer: Principles & Practice of Oncology (3rd Edition)(1989) (pp. 1843-1847). In less than 5 percent of CLL cases, the tumorcells have a T-cell phenotype. Notwithstanding these classifications,however, the pathological impairment of normal hematopoiesis is thehallmark of all leukemias.

Multiple myeloma (MM) is a cancer of plasma cells in the bone marrow.Normally, plasma cells produce antibodies and play a key role in immunefunction. However, uncontrolled growth of these cells leads to bone painand fractures, anemia, infections, and other complications. Multiplemyeloma is the second most common hematological malignancy, although theexact causes of multiple myeloma remain unknown. Multiple myeloma causeshigh levels of proteins in the blood, urine, and organs, including butnot limited to M-protein and other immunoglobulins (antibodies),albumin, and beta-2-microglobulin. M-protein, short for monoclonalprotein, also known as paraprotein, is a particularly abnormal proteinproduced by the myeloma plasma cells and can be found in the blood orurine of almost all patients with multiple myeloma.

Skeletal symptoms, including bone pain, are among the most clinicallysignificant symptoms of multiple myeloma. Malignant plasma cells releaseosteoclast stimulating factors (including IL-1, IL-6 and TNF) whichcause calcium to be leached from bones causing lytic lesions;hypercalcemia is another symptom. The osteoclast stimulating factors,also referred to as cytokines, may prevent apoptosis, or death ofmyeloma cells. Fifty percent of patients have radiologically detectablemyeloma-related skeletal lesions at diagnosis. Other common clinicalsymptoms for multiple myeloma include polyneuropathy, anemia,hyperviscosity, infections, and renal insufficiency.

Bone marrow stromal cells are well known to support multiple myelomadisease progression and resistance to chemotherapy. Disrupting theinteractions between multiple myeloma cells and stromal cells is anadditional target of multiple myeloma chemotherapy.

Myelodysplastic syndrome (MDS) refers to a diverse group ofhematopoietic stem cell disorders. MDS is characterized by a cellularmarrow with impaired morphology and maturation (dysmyelopoiesis),peripheral blood cytopenias, and a variable risk of progression to acuteleukemia, resulting from ineffective blood cell production. See TheMerck Manual 953 (17th ed. 1999) and List et al., 1990, J. Clin. Oncol.8:1424. The treatment of MDS using immunomodulatory compounds isdescribed in U.S. Patent Publication No. 2004/0220144, the entirety ofwhich is hereby incorporated by reference.

Solid tumors are abnormal masses of tissue that may, but usually do notcontain cysts or liquid areas. Solid tumors may be benign (not cancer),or malignant (cancer). Different types of solid tumors are named for thetype of cells that form them. Examples of types solid tumors include,but are not limited to malignant melanoma, adrenal carcinoma, breastcarcinoma, renal cell cancer, carcinoma of the pancreas, non-small-celllung carcinoma (NSCLC) and carcinoma of unknown primary. Drugs commonlyadministered to patients with various types or stages of solid tumorsinclude, but are not limited to, celebrex, etoposide, cyclophosphamide,docetaxel, apecitabine, IFN, tamoxifen, IL-2, GM-CSF, or a combinationthereof.

While patients who achieve a complete remission after initial therapyhave a good chance for cure, less than 10% of those who do not respondor relapse achieve a cure or a response lasting longer than 3 years. SeeCerny T, et al., Ann Oncol 2002; 13 Suppl 4:211-216. Rituximab is knownto deplete normal host B cells. See M. Aklilu et al., Annals of Oncology15:1109-1114, 2004. The long-term immunologic effects of B celldepletion with rituximab and the characteristics of the reconstituting Bcell pool in lymphoma patients are not well defined, despite thewidespread usage of this therapy. See Jennifer H. Anolik et al.,Clinical Immunology, vol. 122, issue 2, February 2007, pages 139-145.

The approach for patients with relapsed or refractory disease reliesheavily on experimental treatments followed by stem celltransplantation, which may not be appropriate for patients with a poorperformance status or advanced age. Therefore, a tremendous demandexists for new methods that can be used to treat patients with NHL.

The link between cancer an altered cellular metabolism has been wellestablished. See Cairns, R. A., et al. Nature Rev., 2011, 11:85-95.Understanding tumor cell metabolism and the associated genetic changesthereof may lead to the identification of improved methods of cancertreatment. Id. For example, tumor cell survival and proliferation viaincreased glucose metabolism has been linked to the PIK3 pathway,whereby mutations in tumor suppressor genes such as PTEN activate tumorcell metabolism. Id. AKT1 (a.k.a., PKB) stimulates glucose metabolismassociated with tumor cell growth by various interactions with PFKFB3,ENTPD5, mTOR and TSC2 (a.k.a., tuberin). Id.

Transcription factors HIF1 and HIF2 are largely responsible for cellularresponse to low oxygen conditions often associated with tumors. Id. Onceactivated, HIF1 promotes tumor cell capacity to carry out glycolysis.Id. Thus, inhibition of HIF1 may slow or reverse tumor cell metabolism.Activation of HIF1 has been linked to PI3K, tumor suppressor proteinssuch as VHL, succinate dehydrogenase (SDH) and fumarate hydratase. Id.The oncogenic transcription factor MYC has also been linked to tumorcell metabolism, specifically glycolysis. Id. MYC also promotes cellproliferation by glutamine metabolic pathways. Id.

AMP-activated protein kinase (AMPK) functions as a metabolic check pointwhich tumor cells must overcome in order to proliferate. Id. Severalmutations have been identified which suppress AMPK signaling in tumorcells. See Shackelford, D. B. & Shaw, R. J., Nature Rev. Cancer, 2009,9: 563-575. STK11 has been identified as a tumor suppressor gene relatedto the role of AMPK. See Cairns, R. A., et al. Nature Rev., 2011,11:85-95.

The transcription factor p53, a tumor suppressor, also has an importantrole in the regulation of cellular metabolism. Id. The loss of p53 intumor cells may be a significant contributor to changes in tumor cellmetabolism to the glycolytic pathway. Id. The OCT1 transcription factor,another potential target for chemotherapeutics, may cooperate with p53in regulating tumor cell metabolism. Id.

Pyruvate kinate M2 (PKM2) promotes changes in cellular metabolism whichconfer metabolic advantages to cancer cells by supporting cellproliferation. Id. For example, lung cancer cells which express PKM2over PKM1 have been found to have such an advantage. Id. In the clinic,PKM2 has been identified as being overexpressed in a number of cancertypes. Id. Thus PKM2 may be a useful biomarker for the early detectionof tumors.

Mutations in isocitrate dehydrogenases IDH1 and IDH2 have been linked totumorigenesis, specifically, in glioblastoma and acute myeloid leukemia.See Mardis, E. R. et al., N. Engl. J. Med., 2009, 361: 1058-1066;Parsons, D. W. et al., Science, 2008, 321: 1807-1812.

The incidence of cancer continues to climb as the general populationages, as new cancers develop, and as susceptible populations (e.g.,people infected with AIDS, the elderly or excessively exposed tosunlight) grow. A tremendous demand therefore exists for new methods,treatments and compositions that can be used to treat patients withcancer including but not limited to those with lymphoma, NHL, multiplemyeloma, AML, leukemias, and solid tumors.

A variety of other diseases and disorders are also associated with, orcharacterized by, undesired angiogenesis. For example, enhanced orunregulated angiogenesis has been implicated in a number of diseases andmedical conditions including, but not limited to, ocular neovasculardiseases, choroidal neovascular diseases, retina neovascular diseases,rubeosis (neovascularization of the angle), viral diseases, geneticdiseases, inflammatory diseases, allergic diseases, fibrosis, arthritisand autoimmune diseases. Examples of such diseases and conditionsinclude, but are not limited to: diabetic retinopathy; retinopathy ofprematurity; corneal graft rejection; neovascular glaucoma; retrolentalfibroplasia; and proliferative vitreoretinopathy.

Accordingly, compounds that can control and/or inhibit unwantedangiogenesis or inhibit the production of certain cytokines, includingTNF-α, may be useful in the treatment and prevention of various diseasesand conditions.

2.2 Inflammatory Diseases

Inflammation plays a fundamental role in host defenses and theprogression of immune-mediated diseases. The inflammatory response isinitiated in response to injury (e.g., trauma, ischemia, and foreignparticles) and infection (e.g., bacterial or viral infection) by acomplex cascade of events, including chemical mediators (e.g., cytokinesand prostaglandins) and inflammatory cells (e.g., leukocytes). Theinflammatory response is characterized by increased blood flow,increased capillary permeability, and the influx of phagocytic cells.These events result in swelling, redness, warmth (altered heatpatterns), and pus formation at the site of injury or infection.

Cytokines and prostaglandins control the inflammatory response, and arereleased in an ordered and self-limiting cascade into the blood oraffected tissues. This release of cytokines and prostaglandins increasesthe blood flow to the area of injury or infection, and may result inredness and warmth. Some of these chemicals cause a leak of fluid intothe tissues, resulting in swelling. This protective process maystimulate nerves and cause pain. These changes, when occurring for alimited period in the relevant area, work to the benefit of the body.

Tumor necrosis factor alpha (TNF-α) is a cytokine that is releasedprimarily by mononuclear phagocytes in response to immunostimulators.TNF-α is capable of enhancing most cellular processes, such asdifferentiation, recruitment, proliferation, and proteolyticdegradation. At low levels, TNF-α confers protection against infectiveagents, tumors, and tissue damage. But TNF-α also has a role in manydiseases. When administered to mammals or humans, TNF-α causes oraggravates inflammation, fever, cardiovascular effects, hemorrhage,coagulation, and acute phase responses similar to those seen duringacute infections and shock states. Enhanced or unregulated TNF-αproduction has been implicated in a number of diseases and medicalconditions, for example, cancers, such as solid tumors and blood-bornetumors; heart disease, such as congestive heart failure; and viral,genetic, inflammatory, allergic, and autoimmune diseases.

Adenosine 3′,5′-cyclic monophosphate (cAMP) also plays a role in manydiseases and conditions, such as but not limited to asthma andinflammation, and other conditions (Lowe and Cheng, Drugs of the Future,17(9), 799-807, 1992). It has been shown that the elevation of cAMP ininflammatory leukocytes inhibits their activation and the subsequentrelease of inflammatory mediators, including TNF-α and NF-κB. Increasedlevels of cAMP also leads to the relaxation of airway smooth muscle.

A delicate well-balanced interplay between the humoral and cellularimmune elements in the inflammatory response enables the elimination ofharmful agents and the initiation of the repair of damaged tissue. Whenthis delicately balanced interplay is disrupted, the inflammatoryresponse may result in considerable damage to normal tissue and may bemore harmful than the original insult that initiated the reaction. Inthese cases of uncontrolled inflammatory responses, clinicalintervention is needed to prevent tissue damage and organ dysfunction.Diseases such as psoriasis, rheumatoid arthritis, osteoarthritis,psoriatic arthritis, Crohn's disease, asthma, allergies or inflammatorybowel disease, are characterized by chronic inflammation. Inflammatorydiseases such as arthritis, related arthritic conditions (e.g.,osteoarthritis, rheumatoid arthritis, and psoriatic arthritis),inflammatory bowel disease (e.g., Crohn's disease and ulcerativecolitis), sepsis, psoriasis, atopic dermatitis, contact dermatitis, andchronic obstructive pulmonary disease, chronic inflammatory pulmonarydiseases are also prevalent and problematic ailments. Enhanced orunregulated TNF-α production plays a central role in the inflammatoryresponse and the administration of their antagonists block chronic andacute responses in animal models of inflammatory disease.

Arthritis is a systemic autoimmune disease that can refer to a group ofconditions involving damage to the joints of the body. There are over100 different forms of arthritis. The most common form is osteoarthritis(degenerative joint disease) and other arthritis forms are rheumatoidarthritis, psoriatic arthritis, and related autoimmune diseases such aslupus and gout. Rheumatoid arthritis is characterized by a chronicinflammation of the joints. Both synovial tissue and fluid are invadedby inflammatory cells which lead to cytokine production. T cells andmonocytes infiltrating the joints display an increased activation ofType 1 and 2 immune response markers.

Psoriatic arthritis is a chronic inflammatory arthritic conditionaffecting the skin, the joints, the insertion sites of tendons,ligaments, and fascia. Gladman, Current Opinion in Rheumatology,“Current concepts in psoriatic arthritis,” 2002, 14:361-366, and Ruddyet al., Rheumatology, vol. 2., chapter 71, page 1071, 6th ed., 2001.Psoriatic arthritis is commonly associated with psoriasis. Id.Approximately 7% of patients with psoriasis develop psoriatic arthritis.The Merck Manual, 448 (17th ed., 1999). Psoriatic arthritis may appearin a variety of clinical patterns. There are five general patterns ofpsoriatic arthritis: arthritis of the distal interphalangeal joints,destructive arthritis, symmetric polyarthritis indistinguishable fromrheumatoid arthritis, asymmetric oligoarthritis, andspondyloarthropathy. Ruddy et al., page 1073. Psoriasis appears toprecede the onset of psoriatic arthritis in 60-80% of patients.Occasionally, arthritis and psoriasis appear simultaneously. Cutaneouseruptions may be preceded by the arthropathy.

Psoriasis is a chronic systemic autoimmune disease that appears on theskin. There are five types of psoriasis: plaque, guttate, inverse,pustular and erythrodermic. The most common form, plaque psoriasis, iscommonly seen as red and white hues of scaly patches appearing on thetop first layer of the epidermis. Some patients, though, have nodermatological symptoms. In plaque psoriasis, skin rapidly accumulatesat these sites, which gives it a silvery-white appearance. Plaquesfrequently occur on the skin of the elbows and knees, but can affect anyarea, including the scalp, palms of hands and soles of feet, andgenitals. In contrast to eczema, psoriasis is more likely to be found onthe outer side of the joint. The disorder is a chronic recurringcondition that varies in severity from minor localized patches tocomplete body coverage. Fingernails and toenails are frequently affected(psoriatic nail dystrophy) and can be seen as an isolated symptom.Psoriasis can also cause inflammation of the joints, which is known aspsoriatic arthritis. In psoriasis, one hypothesis is that T cells becomeactive, migrate to the dermis and trigger the release of cytokines,TNF-α in particular, which causes inflammation and the rapidproliferation of keratinocytes.

2.3 Compounds

A number of studies have been conducted with the aim of providingcompounds that can safely and effectively be used to treat diseasesassociated with abnormal production of TNF-α. See, e.g., Marriott, J.B., et al., Expert Opin. Biol. Ther., 2001, 1(4): 1-8; G. W. Muller, etal., J. Med Chem., 1996, 39(17): 3238-3240; and G. W. Muller, et al.,Bioorg & Med Chem Lett., 1998, 8: 2669-2674. Some studies have focusedon a group of compounds selected for their capacity to potently inhibitTNF-α production by LPS stimulated PBMC. L. G. Corral, et al., Ann.Rheum. Dis., 1999, 58:(Suppl I) 1107-1113. These compounds show not onlypotent inhibition of TNF-α but also marked inhibition of LPS inducedmonocyte IL1ß and IL12 production. LPS induced IL6 is also inhibited bysuch compounds, albeit partially. These compounds are potent stimulatorsof LPS induced IL10. Id.

Compounds for the methods provided herein include, but are not limitedto, the substituted 2-(2,6-dioxopiperidin-3-yl) phthalimides andsubstituted 2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoles described inU.S. Pat. Nos. 6,281,230 and 6,316,471, both to G. W. Muller, et al.Still other specific compounds disclosed herein belong to a class ofisoindole-imides disclosed in U.S. Pat. Nos. 6,395,754, 6,555,554,7,091,353, U.S. patent publication no. 2004/0029832, and InternationalPublication No. WO 98/54170, each of which is incorporated herein byreference. Thalidomide, lenalidomide and pomalidomide have shownremarkable responses in patients with multiple myeloma, lymphoma andother hematological diseases such as myelodysplastic syndrome. SeeGalustian C, et al., Expert Opin Pharmacother., 2009, 10:125-133. Thesedrugs display a broad spectrum of activity, including anti-angiogenicproperties, modulation of pro-inflammatory cytokines, co-stimulation ofT cells, increased NK cell toxicity, direct anti-tumor effects andmodulation of stem cell differentiation.

For example, thalidomide and lenalidomide have emerged as importantoptions for the treatment of multiple myeloma in newly diagnosedpatients, in patients with advanced disease who have failed chemotherapyor transplantation, and in patients with relapsed or refractory multiplemyeloma. Lenalidomide in combination with dexamethasone has beenapproved for the treatment of patients with multiple myeloma who havereceived at least one prior therapy. Pomalidomide may also beadministered in combination with dexamethasone. U.S. Patent PublicationNo. 2004/0029832 A1, the disclosure of which is hereby incorporated inits entirety, discloses the treatment of multiple myeloma.

Another compound provided herein is3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione(“Compound B”), which has the following structure:

or an enantiomer or a mixture of enantiomers thereof; or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,clathrate, or polymorph thereof.

Compound B can be prepared according to the methods described in theExamples provided herein or as described in U.S. Pat. No. 7,635,700, thedisclosure of which is incorporated herein by reference in its entirety.The compound can be also synthesized according to other methods apparentto those of skill in the art based upon the teaching herein. In certainembodiments, Compound B is in a crystalline form described in U.S.Provisional Pat. App. No. 61/451,806, filed Mar. 11, 2011, which isincorporated herein by reference in its entirety. In some embodiments,the hydrochloride salt of Compound B is used in the methods providedherein. Methods of treating, preventing and/or managing cancers andother diseases using Compound B are described in U.S. Provisional Pat.App. No. 61/451,995, filed Mar. 11, 2011, which is incorporated hereinby reference in its entirety.

2.4 Cereblon

The protein Cereblon (CRBN) is a 442-amino acid protein conserved fromplant to human. In humans, the CRBN gene has been identified as acandidate gene of an autosomal recessive nonsyndromic mental retardation(ARNSMR). See Higgins, J. J. et al., Neurology, 2004, 63:1927-1931. CRBNwas initially characterized as an RGS-containing novel protein thatinteracted with a calcium-activated potassium channel protein (SLO1) inthe rat brain, and was later shown to interact with a voltage-gatedchloride channel (CIC-2) in the retina with AMPK7 and DDB1. See Jo, S.et al., J. Neurochem, 2005, 94:1212-1224; Hohberger B. et al., FEBSLett, 2009, 583:633-637; Angers S. et al., Nature, 2006, 443:590-593.DDB1 was originally identified as a nucleotide excision repair proteinthat associates with damaged DNA binding protein 2 (DDB2). Its defectiveactivity causes the repair defect in the patients with xerodermapigmentosum complementation group E (XPE). DDB1 also appears to functionas a component of numerous distinct DCX (DDB1-CUL4-X-box) E3ubiquitin-protein ligase complexes which mediate the ubiquitination andsubsequent proteasomal degradation of target proteins. CRBN has alsobeen identified as a target for the development of therapeutic agentsfor diseases of the cerebral cortex. See WO 2010/137547 A1.

Cereblon has recently been identified as a key molecular target thatbinds to thalidomide to cause birth defects. See Ito, T. et al.,Science, 2010, 327:1345-1350. DDB1 was found to interact with CRBN and,thus, was indirectly associated with thalidomide. Moreover, thalidomidewas able to inhibit auto-ubiquitination of CRBN in vitro, suggestingthat thalidomide is an E3 ubiquitin-ligase inhibitor. Id. Importantly,this activity was inhibited by thalidomide in wild-type cells, but notin cells with mutated CRBN binding sites that prevent thalidomidebinding. Id. The thalidomide binding site was mapped to a highlyconserved C-terminal 104 amino acid region in CRBN. Id. Individual pointmutants in CRBN, Y384A and W386A were both defective for thalidomidebinding, with the double point mutant having the lowestthalidomide-binding activity. Id. A link between CRBN and theteratogenic effect of thalidomide was confirmed in animal models ofzebra-fish and chick embryos. Id.

Whether binding to CRBN, the CRBN E3 ubiquitin-ligase complex, or one ormore substrates of CRBN, is required for the beneficial effects ofthalidomide and other drugs is yet to be established. Understandingthese interactions with thalidomide and other drug targets will allowthe definition of the molecular mechanisms of efficacy and/or toxicityand may lead to drugs with improved efficacy and toxicity profiles.

2.5 Methods of Treating Cancer

Current cancer therapy may involve surgery, chemotherapy, hormonaltherapy and/or radiation treatment to eradicate neoplastic cells in apatient (see, for example, Stockdale, 1998, Medicine, vol. 3, Rubensteinand Federman, eds., Chapter 12, Section IV). Recently, cancer therapycould also involve biological therapy or immunotherapy. All of theseapproaches may pose significant drawbacks for the patient. Surgery, forexample, may be contraindicated due to the health of a patient or may beunacceptable to the patient. Additionally, surgery may not completelyremove neoplastic tissue. Radiation therapy is only effective when theneoplastic tissue exhibits a higher sensitivity to radiation than normaltissue. Radiation therapy can also often elicit serious side effects.Hormonal therapy is rarely given as a single agent. Although hormonaltherapy can be effective, it is often used to prevent or delayrecurrence of cancer after other treatments have removed the majority ofcancer cells. Certain biological and other therapies are limited innumber and may produce side effects such as rashes or swellings,flu-like symptoms, including fever, chills and fatigue, digestive tractproblems or allergic reactions.

With respect to chemotherapy, there are a variety of chemotherapeuticagents available for treatment of cancer. A number of cancerchemotherapeutics act by inhibiting DNA synthesis, either directly orindirectly by inhibiting the biosynthesis of deoxyribonucleotidetriphosphate precursors, to prevent DNA replication and concomitant celldivision. Gilman et al., Goodman and Gilman's: The Pharmacological Basisof Therapeutics, Tenth Ed. (McGraw Hill, New York).

Despite availability of a variety of chemotherapeutic agents,chemotherapy has many drawbacks. Stockdale, Medicine, vol. 3, Rubensteinand Federman, eds., ch. 12, sect. 10, 1998. Almost all chemotherapeuticagents are toxic, and chemotherapy causes significant and oftendangerous side effects including severe nausea, bone marrow depression,and immunosuppression. Additionally, even with administration ofcombinations of chemotherapeutic agents, many tumor cells are resistantor develop resistance to the chemotherapeutic agents. In fact, thosecells resistant to the particular chemotherapeutic agents used in thetreatment protocol often prove to be resistant to other drugs, even ifthose agents act by different mechanism from those of the drugs used inthe specific treatment. This phenomenon is referred to as multidrugresistance. Because of the drug resistance, many cancers proverefractory to standard chemotherapeutic treatment protocols.

Other diseases or conditions associated with, or characterized by,undesired angiogenesis are also difficult to treat. However, somecompounds such as protamine, hepain and steroids have been proposed tobe useful in the treatment of certain specific diseases associated with,or characterized by, undesired angiogenesis. Taylor et al., Nature297:307 (1982); Folkman et al., Science 221:719 (1983); and U.S. Pat.Nos. 5,001,116 and 4,994,443. Thalidomide and certain thalidomidederivatives based on their multiple activities have also been proposedfor the treatment of such diseases and conditions. U.S. Pat. Nos.5,593,990, 5,629,327, 5,712,291, 6,071,948 and 6,114,355 to D'Amato.

Still, there is a significant need for safe and effective methods oftreating, preventing and managing cancer and other diseases that arerefractory to standard treatments, such as surgery, radiation therapy,chemotherapy and hormonal therapy, while reducing or avoiding thetoxicities and/or side effects associated with the conventionaltherapies.

2.6 Methods of Treating Inflammatory Diseases

Current treatments for inflammatory diseases and disorders involvesymptomatic medications and immunosuppressive agents to controlsymptoms. For example, nonsteroidal anti-inflammatory drugs (NSAIDs)such as aspirin, ibuprofen, fenoprofen, naproxen, tolmetin, sulindac,meclofenamate sodium, piroxicam, flurbiprofen, diclofenac, oxaprozin,nabumetone, etodolac, and ketoprofen have analgesic andanti-inflammatory effects. However, NSAIDs are believed not to becapable of altering progression of the disease. (Tierney et al. (eds),Current Medical Diagnosis & Treatment, 37 ed., Appleton & Lange (1998),p 793). Moreover, NSAIDs frequently cause gastrointestinal side effects,affect the lower intestinal tract causing perforation or aggravatinginflammatory bowel disease, produce renal toxicity, and prolong bleedingtime. Corticosteroids are another class of drugs that are commonly usedto control inflammatory symptoms. Corticosteroids, like NSAIDs, do notalter the natural progression of the disease, and thus, clinicalmanifestations of active disease commonly reappear when the drug isdiscontinued. The serious problem of untoward reactions resulting fromprolonged corticosteroid therapy (e.g., osteoporosis, increased risk ofinfection, increased appetite, hypertension, edema, peptic ulcers,psychoses) greatly limits its long-term use.

Low doses of immunosuppressive agents such as cytotoxic agents may beused for the treatment of inflammatory disorders. For example, sometreatments for psoriasis and arthritis are based on disease-modifyinganti-rheumatic drugs (DMARDs such as cyclosporine A and methotrexate),anti-inflammatory agents (TNF-α inhibitors such as etanercept), andanalgesics.

New treatments for inflammatory and autoimmune disorders are constantlybeing sought. In particular, any new treatment that reduces the dosageand/or frequency of administration of agents currently being used, or iscapable of making a currently used treatment more effective isconstantly being sought.

3. SUMMARY OF THE INVENTION

Provided herein are uses of the protein cereblon (CRBN) as a predictorof clinical sensitivity to cancer and inflammatory diseases, and patientresponse to treatment with the compounds provided herein. In certainembodiments, the compounds provided herein bind directly to CRBN-DDB1.

Also provided herein are methods for the treatment or management ofcancer and inflammatory diseases using CRBN as a predictive orprognostic factor for the compounds provided herein. In certainembodiments, provided herein are methods for screening or identifyingcancer patients, e.g., multiple myeloma, DLBCL, mantle cell lymphoma,follicular lymphoma, acute myeloblastic leukemia, chronic lymphocyticleukemia, and/or MDS patients, for treatment with thalidomide,lenalidomide and/or pomalidomide, using CRBN levels as a predictive orprognostic factor. In some embodiments, provided herein are methods forselecting patients having a higher response rate to therapy withthalidomide, lenalidomide and/or pomalidomide, using CRBN levels as apredictive or prognostic factor.

In one embodiment, provided herein is a method of predicting patientresponse to treatment of cancer or an inflammatory disease withthalidomide, lenalidomide and/or pomalidomide, the method comprisingobtaining biological material from the patient, and measuring thepresence or absence of CRBN.

In one embodiment, the mRNA or protein is purified from the tumor andthe presence or absence of a biomarker is measured by gene or proteinexpression analysis. In certain embodiments, the presence or absence ofa biomarker is measured by quantitative real-time PCR (QRT-PCR),microarray, flow cytometry or immunofluorescence. In other embodiments,the presence or absence of a biomarker is measured by enzyme-linkedimmunosorbent assay-based methodologies (ELISA) or other similar methodsknown in the art. Biomarkers associated with non-Hodgkin's lymphomas aredescribed, for example, in U.S. Patent Publication No. 2011/0223157, theentirety of which is incorporated by reference in its entirety.

In another embodiment, provided herein is a method of predicting patientresponse to treatment in a cancer patient, the method comprisingobtaining cancer cells from the patient, culturing the cells in thepresence or absence of a compound provided herein, purifying protein orRNA from the cultured cells, and measuring the presence or absence of abiomarker by, e.g., protein or gene expression analysis. The expressionmonitored may be, for example, mRNA expression or protein expression. Inone embodiment, the cancer patient is a lymphoma, leukemia, multiplemyeloma, solid tumor, non-Hodgkin's lymphoma, DLBCL, mantle celllymphoma, follicular lymphoma, acute myeloblastic leukemia, chroniclymphocytic leukemia, MDS or melanoma patient.

In another embodiment, provided herein is a method of monitoring tumorresponse to compound (e.g., drug) treatment in a cancer patient. Themethod comprises obtaining a biological sample from the patient,measuring the expression of a biomarker in the biological sample,administering one or more compounds (e.g., drugs) to the patient,thereafter obtaining a second biological sample from the patient,measuring biomarker expression in the second biological sample, andcomparing the levels of expression, where an increased level ofbiomarker expression after treatment indicates the likelihood of aneffective tumor response. In one embodiment, the cancer patient is alymphoma, leukemia, multiple myeloma, solid tumor, non-Hodgkin'slymphoma, DLBCL, mantle cell lymphoma, follicular lymphoma, acutemyeloblastic leukemia, chronic lymphocytic leukemia, MDS or melanomapatient.

In one embodiment, a decreased level of biomarker expression aftertreatment indicates the likelihood of effective tumor response. Thebiomarker expression monitored can be, for example, mRNA expression orprotein expression. The expression in the treated sample can increase,for example, by about 1.5×, 2.0×, 3×, 5×, or more. In one embodiment,the tumor is a lymphoma, leukemia, multiple myeloma, solid tumor,non-Hodgkin's lymphoma, DLBCL or melanoma.

In another embodiment, provided herein is a method of predicting thesensitivity to compound (e.g., drug) treatment in a cancer patient,specifically, a multiple myeloma or non-Hodgkin's lymphoma patient(e.g., DLBCL). The method comprises obtaining a biological sample fromthe patient, optionally isolating or purifying mRNA from the biologicalsample, amplifying the mRNA transcripts by, e.g., RT-PCR, where a higherbaseline level of a specific biomarker indicates a higher likelihoodthat the cancer will be sensitive to treatment with a compound (e.g.,drug). In certain embodiments, the biomarker is a gene or proteinassociated with multiple myeloma or non-Hodgkin's lymphoma (e.g.,DLBCL). In one embodiment, the genes are selected from the groupconsisting of DDB1, DDB2, GSK3B, CUL4A, CUL4B, XBP-1, FAS1, RANBP6,DUS3L, PHGDH, AMPK, IRF4 and NFκB.

In one embodiment, identifying a patient having lymphoma, leukemia,multiple myeloma, solid tumor, non-Hodgkin's lymphoma, DLBCL, mantlecell lymphoma, follicular lymphoma, acute myeloblastic leukemia, chroniclymphocytic leukemia, MDS or melanoma sensitive to treatment withthalidomide, lenalidomide, pomalidomide and/or3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione,comprises identifying a gene or protein associated with CRBN. In oneembodiment, the gene or protein associated with CRBN is selected fromthe group consisting of DDB1, DDB2, GSK3B, CUL4A, CUL4B, XBP-1, FAS1,RANBP6, DUS3L, PHGDH, AMPK, IRF4 and NFκB.

In one embodiment, identifying a patient having lymphoma, leukemia,multiple myeloma, a solid tumor, non-Hodgkin's lymphoma, DLBCL ormelanoma sensitive to treatment with thalidomide, lenalidomide,pomalidomide and/or3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dionecomprises measuring the level of CRBN activity in the patient. Inanother embodiment, measuring the level of CRBN activity in the patientcomprises measuring DDB1, DDB2, GSK3B, CUL4A, CUL4B, XBP-1, FAS1,RANBP6, DUS3L, PHGDH, AMPK, IRF4 and/or NFκB in cells obtained from thepatient.

In still other embodiments, provided herein are methods of predictingthe sensitivity to compound (e.g., drug) treatment in a patient having adisease or disorder selected from systemic lupus erythematosus,ANCA-induced vasculitis, glomerulonephritis, acute Wegener'sgranulomatosis, Myasthenia Gravis, Sjogren Syndrome, anti-phospholipidsyndrome, rheumatoid arthritis and fibrotic conditions such as systemicsclerosis. The method comprises obtaining a biological sample from thepatient, optionally isolating or purifying mRNA from the biologicalsample, amplifying the mRNA transcripts by, e.g., RT-PCR, where a higherbaseline level of a specific biomarker indicates a higher likelihoodthat the disease or disorder will be sensitive to treatment with acompound (e.g., drug). In certain embodiments, the biomarker is a geneor protein selected from the group consisting of DDB1, DDB2, GSK3B,CUL4A, CUL4B, XBP-1, FAS1, RANBP6, DUS3L, PHGDH, AMPK, IRF4 and NFκB.

In one embodiment, identifying a patient having systemic lupuserythematosus, ANCA-induced vasculitis, glomerulonephritis, acuteWegener's granulomatosis, Myasthenia Gravis, Sjogren Syndrome,anti-phospholipid syndrome, rheumatoid arthritis or systemic sclerosisand sensitive to treatment with thalidomide, lenalidomide, pomalidomideand/or3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione,comprises identifying a gene or protein associated with CRBN. In oneembodiment, the gene or protein associated with CRBN is selected fromthe group consisting of DDB1, DDB2, GSK3B, CUL4A, CUL4B, XBP-1, FAS1,RANBP6, DUS3L, PHGDH, AMPK, IRF4 and NFκB.

In one embodiment, identifying a patient having systemic lupuserythematosus, ANCA-induced vasculitis, glomerulonephritis, acuteWegener's granulomatosis, Myasthenia Gravis, Sjogren Syndrome,anti-phospholipid syndrome, rheumatoid arthritis or systemic sclerosissensitive to treatment with thalidomide, lenalidomide, pomalidomideand/or3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dionecomprises measuring the level of CRBN activity in the patient. Inanother embodiment, measuring the level of CRBN activity in the patientcomprises measuring DDB1, DDB2, GSK3B, CUL4A, CUL4B, XBP-1, FAS1,RANBP6, DUS3L, PHGDH, AMPK, IRF4 and/or NFκB in cells obtained from thepatient.

In one embodiment, the compound is thalidomide.

In another embodiment, the compound is lenalidomide.

In another embodiment, the compound is pomalidomide.

In another embodiment, the compound is3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione, oran enantiomer thereof, or a pharmaceutically acceptable salt, polymorph,solvate or hydrate thereof.

Also provided herein are kits useful for predicting the likelihood of aneffective lymphoma, leukemia, multiple myeloma, a solid tumor,non-Hodgkin's lymphoma, diffuse large B-cell lymphoma mantle celllymphoma, follicular lymphoma, acute myeloblastic leukemia, chroniclymphocytic leukemia, MDS or melanoma treatment or for monitoring theeffectiveness of a treatment with one or more compounds (e.g., drugs).The kit comprises a solid support, and a means for detecting the proteinexpression of at least one biomarker in a biological sample. Such a kitmay employ, for example, a dipstick, a membrane, a chip, a disk, a teststrip, a filter, a microsphere, a slide, a multiwell plate, or anoptical fiber. The solid support of the kit can be, for example, aplastic, silicon, a metal, a resin, glass, a membrane, a particle, aprecipitate, a gel, a polymer, a sheet, a sphere, a polysaccharide, acapillary, a film, a plate, or a slide. The biological sample can be,for example, a cell culture, a cell line, a tissue, an oral tissue,gastrointestinal tissue, an organ, an organelle, a biological fluid, ablood sample, a urine sample, or a skin sample. The biological samplecan be, for example, a lymph node biopsy, a bone marrow biopsy, or asample of peripheral blood tumor cells.

In another embodiment, the kit comprises a solid support, nucleic acidscontacting the support, where the nucleic acids are complementary to atleast 20, 50, 100, 200, 350, or more bases of mRNA, and a means fordetecting the expression of the mRNA in a biological sample.

In certain embodiments, the kits provided herein employ means fordetecting the expression of a biomarker by quantitative real-time PCR(QRT-PCR), microarray, flow cytometry or immunofluorescence. In otherembodiments, the expression of the biomarker is measured by ELISA-basedmethodologies or other similar methods known in the art.

In still other embodiments, the kits provided herein are useful forpredicting the likelihood of an effective treatment of a disease ordisorder selected from systemic lupus erythematosus, ANCA-inducedvasculitis, glomerulonephritis, acute Wegener's granulomatosis,Myasthenia Gravis, Sjogren Syndrome, anti-phospholipid syndrome,rheumatoid arthritis and fibrotic conditions such as systemic sclerosis.

In addition to the methods described above, a compound provided hereinis administered in combination with a therapy conventionally used totreat, prevent or manage a disease or disorder described herein.Examples of such conventional therapies include, but are not limited to,surgery, chemotherapy, radiation therapy, hormonal therapy, biologicaltherapy and immunotherapy.

Also provided herein are pharmaceutical compositions, single unit dosageforms, dosing regimens and kits which comprise a compound providedherein, or a pharmaceutically acceptable salt, solvate, hydrate,stereoisomer, clathrate, or prodrug thereof, and a second, oradditional, active agent. Second active agents include specificcombinations, or “cocktails,” of drugs.

4. BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A & 1B: Confirmation of CRBN knockdown by siRNAs in H929 and U266multiple myeloma cells.

FIGS. 2A-2C: Knockdown of CRBN abrogated G1 arrest induced bylenalidomide (“Len”), pomalidomide (“Pom”) and Compound B.

FIG. 3A: CRBN knockdown in U266B1 cells confirmed by RT-PCR.

FIG. 3B: CRBN knockdown abrogates lenalidomide and pomalidomide effecton cell cycle in U266 cells.

FIGS. 3C & 3D: Knockdown of CRBN prevents increase of p21^(WAF1) inlenalidomide and pomalidomide treated U266 cells as detected by RT-PCRand Western blot analysis.

FIGS. 4A-4D: CRBN knockdown abrogates drug effect on phosphorylation ofpRb and IRF-4 in H929 cells.

FIGS. 5A-5C: Cell cycle and gene expression profiles in U266B1 cellstransfected with CRBN-siRNA.

FIGS. 6A-6D: DDB1 knockdown only partially affected cell cycle delayinduced by lenalidomide and pomalidomide in U266 cells.

FIGS. 7A & 7B: Lenalidomide and pomalidomide decrease total K48-linkedpolyubiquitination but not K-63-linked ubiquitination in H929.

FIG. 8: 1 hour up-regulated ubiquitination, no MG132.

FIG. 9: 4 hour up-regulated ubiquitination, no MG132.

FIG. 10: 1 hour up-regulated ubiquitination, MG132.

FIG. 11: 4 hour up-regulated ubiquitination, MG132.

FIGS. 12A-12D: Effect of CRBN knockdown on TNFα and IL-2 levels in Tcells.

FIGS. 13A-13C: Effect of CUL4A and CUL4A knockdown on TNFα and IL-2levels in T cells.

FIG. 14: Antiproliferative activity of lenalidomide vs. CRBN expressionin DLBCL cells.

FIG. 15A: Map of CRBN_034 clone. FIG. 15A discloses “6×His” as SEQ IDNO: 230.

FIG. 15B: Map of DDB1_004 clone.

FIG. 16A: Antiproliferative activity of lenalidomide in CRBN-sensitivemyeloma cells.

FIG. 16B: Antiproliferative activity of pomalidomide in CRBN-sensitivemyeloma cells.

FIG. 16C: Antiproliferative activity of Compound B in CRBN-sensitivemyeloma cells.

FIG. 17: Peptides regulated by lenalidomide (“Len”) and pomalidomide(“Pom”) without MG132 in 1 hour ubiquitination experiments.

FIG. 18: Peptides regulated by lenalidomide (“Len”) and pomalidomide(“Pom”) with MG132 in 1 hour ubiquitination experiments.

FIG. 19: Peptides regulated by lenalidomide (“Len”) and pomalidomide(“Pom”) without MG132 in 4 hour ubiquitination experiments.

FIG. 20: Peptides regulated by lenalidomide (“Len”) and pomalidomide(“Pom”) with MG132 in 4 hour ubiquitination experiments.

FIG. 21: Table of common peptides (SEQ ID NOS 35, 16 and 231,respectively, in order of appearance) between lenalidomide (“Len”) andpomalidomide (“Pom”) in ubiquitination experiments.

FIG. 22: Table of common hits in multiple lenalidomide (“Len”) andpomalidomide (“Pom”) ubiquitination experiments. FIG. 22 discloses SEQID NOS 232-236, 35, 237-238, 150, 92, 239, 55, 240 and 148,respectively, in order of appearance.

FIG. 23: Ubiscan data for lenalidomide (“Len” or “Rev”), pomalidomide(“Pom”), and Compound B. FIG. 23 discloses SEQ ID NOS 32, 113, 158,161-162, 47, 51, 93-95, 104, 193, 210-211, 241-250 and 196,respectively, in order of appearance.

FIG. 24A: ABC-DLBCL signature genes.

FIG. 24B: NF-κB activity and IRF4 activity in DLBCL cells.

FIG. 24C: “ABC Scores” of DLBCL cell lines.

FIG. 25: Lenalidomide inhibits proliferation of ABC-DLBCL cells invitro.

FIG. 26: Lenalidomide treatment also induced apoptosis of sensitive celllines such as OCI-Ly10.

FIGS. 27A-27C: Effect of Lenalidomide on IRF4 expression in ABC-DLBCLcell lines.

FIG. 28: Mutation analysis of CARD11 coiled-coil domain 1 in DLBCL celllines. FIG. 28 discloses SEQ ID NOS 251, 252, 252, 252, 252, 252, 252,252, 252, 252, 252, 252, 252 and 252, respectively, in order ofappearance.

FIGS. 29A-29C: Lenalidomide inhibits activation of CARD11-Bcl-10-MALT1complex in sensitive DLBCL cell lines.

FIGS. 30A-30C: Lenalidomide inhibits NF-κB activity in ABC-DLBCL cells.

FIGS. 31A-31D: Alteration of IRF4 expression in ABC-DLBCL cells affectscell sensitivity to lenalidomide.

FIGS. 32A-32D: Downregulation of IRF4, NF-κB and proliferation bylenalidomide requires the presence of cereblon.

FIGS. 33A-33B: Lenalidomide inhibits tumor growth in mouse xenograftmodel with OCI-Ly10 ABC-DLBCL.

FIGS. 34A-34B: “ABC scores” and baseline IRF4/CRBN levels correlatelenalidomide sensitivity of DLBCL cells.

FIG. 35: Alignment between heavy chain amino acid sequences ofantibodies CGN-6-1-11 (top; SEQ ID NO:5) and CGN-6-4-5 (bottom; SEQ IDNO:9).

FIG. 36: Alignment between light chain amino acid sequences ofantibodies CGN-6-1-11 (top; SEQ ID NO:7) and CGN-6-4-5 (bottom; SEQ IDNO:11).

FIGS. 37A & 37B: Confocal immunofluorescent analysis of DF15 (leftpanel) and DF15R cells (right panel) using 1 μg/ml CGN-6-4-5 antibody(green) (A) or CGN-6-4-5 antibody/CRBN blocking peptide mix (1:5 excessratio) (B). Nuclear staining performed with Dapi (blue).

FIG. 38: Immunoblot with myeloma cells containing endogenous CRBN(DF15), DF15R with no CRBN and HEK293 cells expressing recombinantflag-tagged CRBN.

FIGS. 39A & 39B: Binding of thalidomide and other compounds to CRBN

5. DETAILED DESCRIPTION OF THE INVENTION

The methods provided herein are based, in part, on the discovery thatcereblon is associated with the anti-proliferative activities of certaindrugs, such as the compounds provided herein. In some embodiments,Cereblon (CRBN) may be utilized as a biomarker to indicate theeffectiveness or progress of a disease treatment with a compoundprovided herein.

Without being bound to a particular theory, CRBN binding may contributeto or even be required for anti-proliferative or other activities ofcertain compounds, such as the compounds provided herein. In certainembodiments, the compounds provided herein bind directly to CRBN-DDB1and/or the CRBN E3 ubiquitin-ligase complex. Mutations in CRBN could beassociated with resistance to the compounds provided herein.

For example, the levels of CRBN were significantly lower in thepomalidomide-resistant cells line DF15R and the lenalidomide-resistantcells, H929 R10-1, H929 R10-2, H929 R10-3, H929 R10-4 and MM1/R comparedto the matched parental lines. Furthermore, an interesting mutation wasfound in CRBN gene of one of the myeloma lines that had acquiredresistance to lenalidomide while in the parental line the CRBN gene waswild type. This mutation mapped to the DDB1 binding domain in CRBN.Thus, in certain embodiments, the sensitivity of a cancer cell, e.g., amyeloma cell, or a patient having cancer, to therapy with a compoundprovided herein is related to CRBN expression.

In relapsed or refractory diffuse large B-cell lymphoma (DLBCL), higherresponses were seen in the activated B-cell-like (ABC) subtype than thegerminal center B-cell-like subtype. As provided herein using DLBCL celllines, it was shown that lenalidomide treatment preferentiallysuppressed proliferation of ABC-DLBCL cells in vitro and delayed tumorgrowth in a human tumor xenograft model, with minimal effect onnon-ABC-DLBCL cells. This tumoricidal effect was associated withdownregulation of interferon regulatory factor 4 (IRF4), a hallmark ofABC-DLBCL cells.

IRF4 inhibition by lenalidomide caused downregulation of B cell receptor(BCR)-dependent NF-κB activation. While IRF4-specific siRNA mimickedeffects of lenalidomide reducing NF-κB activation, IRF4 overexpressionenhanced NF-κB activation and conferred resistance to lenalidomide.Furthermore, lenalidomide-induced IRF4 downregulation required theexpression of CRBN. Without being bound to a particular theory, thesedata show that lenalidomide may have direct antitumor activity againstDLBCL cells, preferentially ABC-DLBCL cells, by blocking IRF4 expressionand the BCR-NF-κB signaling pathway in a CRBN-dependent manner.

It has been proposed that CRBN protein functions as a substrate receptorfor Cul4-E3-ligase complexes through its interaction with DDB1. Asprovided herein, whether in vivo ubiquitination is associated with drugresponses in multiple myeloma cells has been investigated. In H929cells, compounds provided herein decrease total K48-linkedpolyubiquitination but not K-63-linked ubiquitination after 30 minutestreatment. At present, nearly two dozen proteins are reported to bedegraded by a Cul4-DDB1 ligase2. Several studies have shownCul4/DDB1-dependent ubiquitination of core histones, DNA repairproteins, cell cycle regulators and key signaling pathways molecules.mTORC1 signaling requires proteasomal function and the involvement ofCUL4-DDB1 ubiquitin E3 ligase. Using CST Ubiscan technology, 162 uniqueubiquitin-peptides were identified which were significantly modulated bythe compounds provided herein after short treatments (1-4 h). Thecorresponding proteins participate in nucleasome and chromatin function,protein-DNA assembly and histone H2A. The relevance of this earlymodification in the mode of action of compounds provided herein, and therelationship with CRBN and CUL4/DDB1 activities are under investigation.

Provided herein are methods for the treatment or management of cancerand inflammatory diseases using CRBN as a predictive or prognosticfactor for the compounds provided herein. In certain embodiments,provided herein are methods for screening or identifying cancerpatients, e.g., lymphoma, leukemia, multiple myeloma, solid tumor,non-Hodgkin's lymphoma, DLBCL, mantle cell lymphoma, follicularlymphoma, acute myeloblastic leukemia, chronic lymphocytic leukemia, MDSor melanoma patients, for treatment with thalidomide, lenalidomideand/or pomalidomide, using CRBN levels as a predictive or prognosticfactor. In some embodiments, provided herein are methods for selectingpatients having a higher response rate to therapy with thalidomide,lenalidomide and/or pomalidomide, using CRBN levels as a predictive orprognostic factor.

In one embodiment, provided herein is a method of predicting patientresponse to treatment of cancer or an inflammatory disease withthalidomide, lenalidomide and/or pomalidomide, the method comprisingobtaining biological material from the patient, and measuring thepresence or absence of CRBN.

In one embodiment, the mRNA or protein is purified from the tumor andthe presence or absence of a biomarker is measured by gene or proteinexpression analysis. In certain embodiments, the presence or absence ofa biomarker is measured by quantitative real-time PCR (QRT-PCR),microarray, flow cytometry or immunofluorescence. In other embodiments,the presence or absence of a biomarker is measured by enzyme-linkedimmunosorbent assay-based methodologies (ELISA) or other similar methodsknown in the art.

In another embodiment, provided herein is a method of predicting thesensitivity to compound (e.g., drug) treatment in a cancer patient, suchas, a multiple myeloma or non-Hodgkin's lymphoma patient. The methodcomprises obtaining a biological sample from the patient, optionallyisolating or purifying mRNA from the biological sample, amplifying themRNA transcripts by, e.g., RT-PCR, where a higher baseline level of aspecific biomarker indicates a higher likelihood that the cancer will besensitive to treatment with a compound (e.g., drug). In certainembodiments, the biomarker is a gene or protein associated with multiplemyeloma or non-Hodgkin's lymphoma (e.g., DLBCL). In one embodiment, thegenes are selected from the group consisting of DDB1, DDB2, GSK3B,CUL4A, CUL4B, XBP-1, FAS1, RANBP6, DUS3L, PHGDH, AMPK, IRF4 and NFκB.

In one embodiment, identifying a patient having lymphoma, leukemia,multiple myeloma, a solid tumor, non-Hodgkin's lymphoma, diffuse largeB-cell lymphoma, mantle cell lymphoma, follicular lymphoma, acutemyeloblastic leukemia, chronic lymphocytic leukemia, MDS or melanomasensitive to treatment with thalidomide, lenalidomide, pomalidomideand/or3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione,comprises identifying a gene or protein associated with CRBN. In oneembodiment, the gene or protein associated with CRBN is selected fromthe group consisting of DDB1, DDB2, GSK3B, CUL4A, CUL4B, XBP-1, FAS1,RANBP6, DUS3L, PHGDH, AMPK, IRF4 and NFκB.

In one embodiment, identifying a patient having lymphoma, leukemia,multiple myeloma, solid tumor, non-Hodgkin's lymphoma, DLBCL, mantlecell lymphoma, follicular lymphoma, acute myeloblastic leukemia, chroniclymphocytic leukemia, MDS or melanoma sensitive to treatment withthalidomide, lenalidomide, pomalidomide and/or3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dionecomprises measuring the level of CRBN activity in the patient. Inanother embodiment, measuring the level of CRBN activity in the patientcomprises measuring DDB1, DDB2, GSK3B, CUL4A, CUL4B, XBP-1, FAS1,RANBP6, DUS3L, PHGDH, AMPK, IRF4 and/or NFκB in cells obtained from thepatient.

In one embodiment, the compound is thalidomide.

In another embodiment, the compound is lenalidomide.

In another embodiment, the compound is pomalidomide.

In another embodiment, the compound is3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione, oran enantiomer thereof, or a pharmaceutically acceptable salt, polymorph,solvate or hydrate thereof.

In one embodiment, the cancer is multiple myeloma.

In another embodiment, the cancer is non-Hodgkin's lymphoma. In oneembodiment, the non-Hodgkin's lymphoma is of the activated B-cellphenotype.

In another embodiment, the cancer is diffuse large B-cell lymphoma. Inone embodiment, the diffuse large B-cell lymphoma is of the activatedB-cell phenotype.

In another embodiment, the cancer is mantle cell lymphoma.

In another embodiment, the cancer is follicular lymphoma.

In another embodiment, the cancer is acute myeloblastic leukemia.

In another embodiment, the cancer is chronic lymphocytic leukemia.

In another embodiment, the cancer is myelodysplastic syndrome.

In another embodiment, the cancer is melanoma.

In still other embodiments, provided herein are methods of predictingthe sensitivity to compound (e.g., drug) treatment in a patient having adisease or disorder selected from systemic lupus erythematosus,ANCA-induced vasculitis, glomerulonephritis, acute Wegener'sgranulomatosis, Myasthenia Gravis, Sjogren Syndrome, anti-phospholipidsyndrome, rheumatoid arthritis and fibrotic conditions such as systemicsclerosis. The method comprises obtaining a biological sample from thepatient, optionally isolating or purifying mRNA from the biologicalsample, amplifying the mRNA transcripts by, e.g., RT-PCR, where a higherbaseline level of a specific biomarker indicates a higher likelihoodthat the disease or disorder will be sensitive to treatment with acompound (e.g., drug). In certain embodiments, the biomarker is a geneor protein selected from the group consisting of DDB1, DDB2, GSK3B,CUL4A, CUL4B, XBP-1, FAS1, RANBP6, DUS3L, PHGDH, AMPK, IRF4 and NFκB.

In one embodiment, identifying a patient having selected from systemiclupus erythematosus, ANCA-induced vasculitis, glomerulonephritis, acuteWegener's granulomatosis, Myasthenia Gravis, Sjogren Syndrome,anti-phospholipid syndrome, rheumatoid arthritis or systemic sclerosissensitive to treatment with thalidomide, lenalidomide, pomalidomideand/or3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dionecomprises identification of a gene or protein associated with CRBN. Inone embodiment, the gene or protein associated with CRBN is selectedfrom the group consisting of DDB1, DDB2, GSK3B, CUL4A, CUL4B, XBP-1,FAS1, RANBP6, DUS3L, PHGDH, AMPK, IRF4 and NFκB.

In one embodiment, identifying a patient having systemic lupuserythematosus, ANCA-induced vasculitis, glomerulonephritis, acuteWegener's granulomatosis, Myasthenia Gravis, Sjogren Syndrome,anti-phospholipid syndrome, rheumatoid arthritis or systemic sclerosissensitive to treatment with thalidomide, lenalidomide, pomalidomideand/or3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dionecomprises measuring the level of CRBN activity in the patient. Inanother embodiment, measuring the level of CRBN activity in the patientcomprises measuring DDB1, DDB2, GSK3B, CUL4A, CUL4B, XBP-1, FAS1,RANBP6, DUS3L, PHGDH, AMPK, IRF4 and/or NFκB in cells obtained from thepatient.

In one embodiment, the compound is thalidomide.

In another embodiment, the compound is lenalidomide.

In another embodiment, the compound is pomalidomide.

In another embodiment, the compound is3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione, oran enantiomer thereof, or a pharmaceutically acceptable salt, polymorph,solvate or hydrate thereof.

Also provided herein are kits useful for predicting the likelihood of aneffective lymphoma, leukemia, multiple myeloma, a solid tumor,non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, mantle celllymphoma, follicular lymphoma, acute myeloblastic leukemia, chroniclymphocytic leukemia, myelodysplastic syndrome or melanoma treatment orfor monitoring the effectiveness of a treatment with one or morecompounds (e.g., drugs). The kit comprises a solid support, and a meansfor detecting the protein expression of at least one biomarker in abiological sample. Such a kit may employ, for example, a dipstick, amembrane, a chip, a disk, a test strip, a filter, a microsphere, aslide, a multiwell plate, or an optical fiber. The solid support of thekit can be, for example, a plastic, silicon, a metal, a resin, glass, amembrane, a particle, a precipitate, a gel, a polymer, a sheet, asphere, a polysaccharide, a capillary, a film, a plate, or a slide. Thebiological sample can be, for example, a cell culture, a cell line, atissue, an oral tissue, gastrointestinal tissue, an organ, an organelle,a biological fluid, a blood sample, a urine sample, or a skin sample.The biological sample can be, for example, a lymph node biopsy, a bonemarrow biopsy, or a sample of peripheral blood tumor cells.

In another embodiment, the kit comprises a solid support, nucleic acidscontacting the support, where the nucleic acids are complementary to atleast 20, 50, 100, 200, 350, or more bases of mRNA, and a means fordetecting the expression of the mRNA in a biological sample.

In certain embodiments, the kits provided herein employ means fordetecting the expression of a biomarker by quantitative real-time PCR(QRT-PCR), microarray, flow cytometry or immunofluorescence. In otherembodiments, the expression of the biomarker is measured by ELISA-basedmethodologies or other similar methods known in the art.

In still other embodiments, the kits provided herein are useful forpredicting the likelihood of an effective treatment of a disease ordisorder selected from systemic lupus erythematosus, ANCA-inducedvasculitis, glomerulonephritis, acute Wegener's granulomatosis,Myasthenia Gravis, Sjogren Syndrome, anti-phospholipid syndrome,rheumatoid arthritis and fibrotic conditions such as systemic sclerosis.

Provided herein is a method of selecting a group of cancer patientsbased on the level of CRBN expression, or the levels of DDB1, DDB2,GSK3B, CUL4A, CUL4B, XBP-1, FAS1, RANBP6, DUS3L, PHGDH, AMPK, IRF4 orNFκB expression within the cancer, for the purposes of predictingclinical response, monitoring clinical response, or monitoring patientcompliance to dosing by thalidomide, lenalidomide, pomalidomide or3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione, astereoisomer thereof, or a pharmaceutically acceptable salt, solvate,hydrate, co-crystal, clathrate, or polymorph thereof; wherein the cancerpatients are selected from multiple myeloma, non-Hodgkin's lymphoma,diffuse large B-cell lymphoma, melanoma and solid tumor patients.

In one embodiment, the cancer patients are multiple myeloma patients.

In one embodiment, cancer patients are non-Hodgkin's lymphoma patients.In one embodiment, the non-Hodgkin's lymphoma is of the activated B-cellphenotype.

In one embodiment, cancer patients are diffuse large B-cell lymphomapatients. In one embodiment, the diffuse large B-cell lymphoma is of theactivated B-cell phenotype.

In one embodiment, method of selecting a group of cancer patients isbased on the level of DDB1 expression within the cancer.

In one embodiment, the method of selecting a group of cancer patients isbased on the level of DDB2 expression within the cancer.

In one embodiment, the method of selecting a group of cancer patients isbased on the level of GSK3B expression within the cancer.

In one embodiment, the method of selecting a group of cancer patients isbased on the level of CUL4A expression within the cancer.

In one embodiment, the method of selecting a group of cancer patients isbased on the level of CUL4B expression within the cancer.

In one embodiment, the method of selecting a group of cancer patients isbased on the level of XBP-1 expression within the cancer.

In one embodiment, the method of selecting a group of cancer patients isbased on the level of FAS1 expression within the cancer.

In one embodiment, the method of selecting a group of cancer patients isbased on the level of RANBP6 expression within the cancer.

In one embodiment, the method of selecting a group of cancer patients isbased on the level of DUS3L expression within the cancer.

In one embodiment, the method of selecting a group of cancer patients isbased on the level of PHGDH expression within the cancer.

In one embodiment, the method of selecting a group of cancer patients isbased on the level of AMPK expression within the cancer.

In one embodiment, the method of selecting a group of cancer patients isbased on the level of IRF4 expression within the cancer.

In one embodiment, the method of selecting a group of cancer patients isbased on the level of NFκB expression within the cancer.

Also provided herein is a method of identifying or monitoring multiplemyeloma patient resistance to thalidomide, lenalidomide, pomalidomide or3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dionetherapy, based on the presence or appearance of mutations within a CRBNgene.

In one embodiment, the mutation with the CRBN gene is asingle-nucleotide polymorphism in the coding region c.745C>CA causing anamino acid change 249D>YD in the protein within the DDB1 binding domainof CRBN.

In another embodiment, provided herein is a method of selecting a groupof patients responsive to treatment with thalidomide, lenalidomide,pomalidomide or3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione, astereoisomer thereof, or a pharmaceutically acceptable salt, solvate,hydrate, co-crystal, clathrate, or polymorph thereof; based on the levelof CRBN expression, or the levels of DDB1, DDB2, GSK3B, CUL4A, CUL4B,XBP-1, FAS1, RANBP6, DUS3L, PHGDH, AMPK, IRF4 or NFκB expression withinthe patient's T cells, B cells, or plasma cells, for the purposes ofpredicting clinical response, monitoring clinical response, ormonitoring patient compliance to dosing by thalidomide, lenalidomide,pomalidomide or3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione, astereoisomer thereof, or a pharmaceutically acceptable salt, solvate,hydrate, co-crystal, clathrate, or polymorph thereof.

Also provided herein is an isolated CRBN antibody, for example,“CRBN70,” as prepared according to Example 6.20 or 6.21 below. In oneembodiment, the antibody is a polyclonal antibody. In anotherembodiment, the antibody is a monoclonal antibody. In some embodiments,the antibody is a rabbit polyclonal antibody. In other embodiments, theantibody is a rabbit monoclonal antibody.

In another embodiment, provided herein is an isolated antibody whichimmunospecifically binds to the epitope having an amino acid sequenceEEFHGRTLHDDDC (SEQ ID:1). In another embodiment, the antibodyimmunospecifically binds to the epitope having an amino acid sequenceEEFHGRTLHDDDC (SEQ ID:1), wherein the peptide is coupled to KeyholeLimpet Hemocyanin (KLH). In one embodiment, the antibody is a polyclonalantibody. In another embodiment, the antibody is a monoclonal antibody.In some embodiments, the antibody is a rabbit polyclonal antibody. Inother embodiments, the antibody is a rabbit monoclonal antibody. Incertain embodiments, the antibody immunospecifically binds peptide 65-76(SEQ ID NO:1) of human CRBN (SEQ ID NO:12).

In certain embodiments, provided herein is an antibody thatimmunospecifically binds CRBN and comprises a heavy chain having theamino acid sequence depicted in SEQ ID NO:5. In other embodiment, theantibody immunospecifically binds CRBN and comprises a light chainhaving the amino acid sequence depicted in SEQ ID NO:7. In someembodiments, the antibody comprises a heavy chain having the amino acidsequence depicted in SEQ ID NO:5 and a light chain having the amino acidsequence depicted in SEQ ID NO:7. In certain embodiments, the antibodyimmunospecifically binds CRBN and comprises a heavy chain having theamino acid sequence depicted in SEQ ID NO:9. In other embodiment, theantibody immunospecifically binds CRBN and comprises a light chainhaving the amino acid sequence depicted in SEQ ID NO:11. In someembodiments, the antibody comprises a heavy chain having the amino acidsequence depicted in SEQ ID NO:9 and a light chain having the amino acidsequence depicted in SEQ ID NO:11. In certain embodiments, the antibodyimmunospecifically binds peptide 65-76 (SEQ ID NO:1) of human CRBN (SEQID NO:12).

Also provided herein is a method of utilizing a CRBN antibody (e.g., arabbit polyclonal or monoclonal antibody CRBN70, or a rabbit polyclonalor monoclonal antibody that binds peptide 65-76 (SEQ ID NO:1) of humanCRBN (SEQ ID NO:12) to measure expression levels of CRBN in patienttumor or host cells, to predict clinical response, monitor clinicalresponse, monitor patient compliance to dosing, or monitor developmentof resistance to therapy with thalidomide, lenalidomide, pomalidomide,or 3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione, astereoisomer thereof, or a pharmaceutically acceptable salt, solvate,hydrate, co-crystal, clathrate, or polymorph thereof. In one embodiment,the CRBN antibody immunospecifically binds to the epitope having anamino acid sequence EEFHGRTLHDDD (residues 1-12 of SEQ ID NO:1). In anembodiment, the CRBN antibody specifically binds to EEFHGRTLHDDD(residues 1-12 of SEQ ID NO:1), which is coupled to Keyhole LimpetHemocyanin (KLH). In one embodiment, the CRBN antibody is a polyclonalantibody, such as a rabbit polyclonal antibody. In another embodiment,the CRBN antibody is a monoclonal antibody, such as a rabbit monoclonalantibody.

5.1 Definitions

As used herein, and unless otherwise specified, the terms “treat,”“treating” and “treatment” refer to an action that occurs while apatient is suffering from the specified cancer, which reduces theseverity of the cancer, or retards or slows the progression of thecancer.

The term “sensitivity” and “sensitive” when made in reference totreatment with compound is a relative term which refers to the degree ofeffectiveness of the compound in lessening or decreasing the progress ofa tumor or the disease being treated. For example, the term “increasedsensitivity” when used in reference to treatment of a cell or tumor inconnection with a compound refers to an increase of, at least a 5%, ormore, in the effectiveness of the tumor treatment.

As used herein, and unless otherwise specified, the term“therapeutically effective amount” of a compound is an amount sufficientto provide a therapeutic benefit in the treatment or management of acancer, or to delay or minimize one or more symptoms associated with thepresence of the cancer. A therapeutically effective amount of a compoundmeans an amount of therapeutic agent, alone or in combination with othertherapies, which provides a therapeutic benefit in the treatment ormanagement of the cancer. The term “therapeutically effective amount”can encompass an amount that improves overall therapy, reduces or avoidssymptoms or causes of cancer, or enhances the therapeutic efficacy ofanother therapeutic agent.

As used herein, an “effective patient tumor response” refers to anyincrease in the therapeutic benefit to the patient. An “effectivepatient tumor response” can be, for example, a 5%, 10%, 25%, 50%, or100% decrease in the rate of progress of the tumor. An “effectivepatient tumor response” can be, for example, a 5%, 10%, 25%, 50%, or100% decrease in the physical symptoms of a cancer. An “effectivepatient tumor response” can also be, for example, a 5%, 10%, 25%, 50%,100%, 200%, or more increase in the response of the patient, as measuredby any suitable means, such as gene expression, cell counts, assayresults, etc.

The term “likelihood” generally refers to an increase in the probabilityof an event. The term “likelihood” when used in reference to theeffectiveness of a patient tumor response generally contemplates anincreased probability that the rate of tumor progress or tumor cellgrowth will decrease. The term “likelihood” when used in reference tothe effectiveness of a patient tumor response can also generally meanthe increase of indicators, such as mRNA or protein expression, that mayevidence an increase in the progress in treating the tumor.

The term “predict” generally means to determine or tell in advance. Whenused to “predict” the effectiveness of a cancer treatment, for example,the term “predict” can mean that the likelihood of the outcome of thecancer treatment can be determined at the outset, before the treatmenthas begun, or before the treatment period has progressed substantially.

The term “monitor,” as used herein, generally refers to the overseeing,supervision, regulation, watching, tracking, or surveillance of anactivity. For example, the term “monitoring the effectiveness of acompound” refers to tracking the effectiveness in treating a cancer in apatient or in a tumor cell culture. Similarly, the “monitoring,” whenused in connection with patient compliance, either individually, or in aclinical trial, refers to the tracking or confirming that the patient isactually taking a drug being tested as prescribed. The monitoring can beperformed, for example, by following the expression of mRNA or proteinbiomarkers.

An improvement in the cancer or cancer-related disease can becharacterized as a complete or partial response. “Complete response”refers to an absence of clinically detectable disease with normalizationof any previously abnormal radiographic studies, bone marrow, andcerebrospinal fluid (CSF) or abnormal monoclonal protein measurements.“Partial response” refers to at least about a 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, or 90% decrease in all measurable tumor burden (i.e., thenumber of malignant cells present in the subject, or the measured bulkof tumor masses or the quantity of abnormal monoclonal protein) in theabsence of new lesions. The term “treatment” contemplates both acomplete and a partial response.

“Tumor,” as used herein, refers to all neoplastic cell growth andproliferation, whether malignant or benign, and all pre-cancerous andcancerous cells and tissues. “Neoplastic,” as used herein, refers to anyform of dysregulated or unregulated cell growth, whether malignant orbenign, resulting in abnormal tissue growth. Thus, “neoplastic cells”include malignant and benign cells having dysregulated or unregulatedcell growth.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include, but are not limitedto, blood-borne tumors (e.g., multiple myeloma, lymphoma and leukemia),and solid tumors.

The term “refractory or resistant” refers to a circumstance wherepatients, even after intensive treatment, have residual cancer cells(e.g., leukemia or lymphoma cells) in their lymphatic system, bloodand/or blood forming tissues (e.g., marrow).

As used herein the terms “polypeptide” and “protein” as usedinterchangeably herein, refer to a polymer of amino acids of three ormore amino acids in a serial array, linked through peptide bonds. Theterm “polypeptide” includes proteins, protein fragments, proteinanalogues, oligopeptides and the like. The term polypeptide as usedherein can also refer to a peptide. The amino acids making up thepolypeptide may be naturally derived, or may be synthetic. Thepolypeptide can be purified from a biological sample.

The term “antibody” is used herein in the broadest sense and coversfully assembled antibodies, antibody fragments which retain the abilityto specifically bind to the antigen (e.g., Fab, F(ab′)2, Fv, and otherfragments), single chain antibodies, diabodies, antibody chimeras,hybrid antibodies, bispecific antibodies, humanized antibodies, and thelike. The term “antibody” covers both polyclonal and monoclonalantibodies.

The term “antibody” and “immunoglobulin” or “Ig” may be usedinterchangeably herein. The terms “antibodies that immunospecificallybind to a CRBN antigen,” “antibodies that immunospecifically bind to aCRBN epitope,” “CRBN antibodies,” “anti-CRBN antibodies” and analogousterms are also used interchangeably herein and refer to antibodies andfragments thereof, that specifically bind to a CRBN polypeptide, such asa CRBN antigen or epitope (e.g., EEFHGRTLHDDD (residues 1-12 of SEQ IDNO:1) or peptide 65-76 human CRBN (SEQ ID NO:12)). The antibodies,including both modified antibodies (i.e., antibodies that comprise amodified IgG (e.g., IgG1) constant domain and unmodified antibodies(i.e., antibodies that do not comprise a modified IgG (e.g., IgG1)constant domain that specifically bind to a CRBN polypeptide. Anantibody or a fragment thereof that immunospecifically binds to a CRBNantigen may be cross-reactive with related antigens. In certainembodiments, an antibody or a fragment thereof that immunospecificallybinds to a CRBN antigen does not cross-react with other antigens. Anantibody or a fragment thereof that immunospecifically binds to a CRBNantigen can be identified, for example, by immunoassays, BIAcore, orother techniques known to those of skill in the art. An antibody or afragment thereof binds specifically to a CRBN antigen when it binds to aCRBN antigen with higher affinity than to any cross-reactive antigen asdetermined using experimental techniques, such as radioimmunoassays(RIA) and enzyme-linked immunosorbent assays (ELISAs). Typically aspecific or selective reaction will be at least twice background signalor noise and more typically more than 10 times background. See, e.g.,Paul, ed., 1989, Fundamental Immunology Second Edition, Raven Press, NewYork at pages 332-336 for a discussion regarding antibody specificity.

Antibodies provided herein include, but are not limited to, syntheticantibodies, monoclonal antibodies, recombinantly produced antibodies,multispecific antibodies (including bi-specific antibodies), humanantibodies, humanized antibodies, chimeric antibodies, intrabodies,single-chain Fvs (scFv) (e.g., including monospecific, bispecific,etc.), camelized antibodies, Fab fragments, F(ab″) fragments,disulfide-linked Fvs (sdFv), anti-idiotypic (anti-Id) antibodies, andepitope-binding fragments of any of the above. In particular, antibodiesprovided herein include immunoglobulin molecules and immunologicallyactive portions of immunoglobulin molecules, i.e., antigen bindingdomains or molecules that contain an antigen-binding site thatimmunospecifically binds to a CRBN antigen (e.g., one or morecomplementarity determining regions (CDRs) of an anti-CRBN antibody).The antibodies provided herein can be of any type (e.g., IgG, IgE, IgM,IgD, IgA and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 andIgA2), or any subclass (e.g., IgG2a and IgG2b) of immunoglobulinmolecule. In some embodiments, the anti-CRBN antibodies are fully human,such as fully human monoclonal CRBN antibodies. In certain embodiments,antibodies provided herein are IgG antibodies, or a class (e.g., humanIgG1 or IgG4) or subclass thereof.

The term “antigen binding domain,” “antigen binding region,” “antigenbinding fragment,” and similar terms refer to that portion of anantibody which comprises the amino acid residues that interact with anantigen and confer on the binding agent its specificity and affinity forthe antigen (e.g., the CDR). The antigen binding region can be derivedfrom any animal species, such as rodents (e.g., rabbit, rat or hamster)and humans. In some embodiments, the antigen binding region will be ofhuman origin.

The term “constant region” or “constant domain” of an antibody refers toa carboxy terminal portion of the light and heavy chain which is notdirectly involved in binding of the antibody to antigen but exhibitsvarious effector function, such as interaction with the Fc receptor. Theterms refer to the portion of an immunoglobulin molecule having a moreconserved amino acid sequence relative to the other portion of theimmunoglobulin, the variable domain, which contains the antigen bindingsite. The constant domain contains the CH1, CH2 and CH3 domains of theheavy chain and the CL domain of the light chain.

The term “epitope” as used herein refers to a localized region on thesurface of an antigen, such as CRBN polypeptide or CRBN polypeptidefragment, that is capable of being bound to one or more antigen bindingregions of an antibody, and that has antigenic or immunogenic activityin an animal, such as a mammal (e.g., a human), that is capable ofeliciting an immune response. An epitope having immunogenic activity isa portion of a polypeptide that elicits an antibody response in ananimal. An epitope having antigenic activity is a portion of apolypeptide to which an antibody immunospecifically binds as determinedby any method well known in the art, for example, by the immunoassaysdescribed herein. Antigenic epitopes need not necessarily beimmunogenic. Epitopes usually consist of chemically active surfacegroupings of molecules such as amino acids or sugar side chains and havespecific three dimensional structural characteristics as well asspecific charge characteristics. A region of a polypeptide contributingto an epitope may be contiguous amino acids of the polypeptide or theepitope may come together from two or more non-contiguous regions of thepolypeptide. The epitope may or may not be a three-dimensional surfacefeature of the antigen. An exemplary epitope of CRBN provided herein isEEFHGRTLHDDD (residues 1-12 of SEQ ID NO:1) or peptide 65-60 of CRBN(SEQ ID NO:13).

The terms “fully human antibody” or “human antibody” are usedinterchangeably herein and refer to an antibody that comprises a humanvariable region and, in some embodiments, a human constant region. Inspecific embodiments, the terms refer to an antibody that comprises avariable region and constant region of human origin. “Fully human”anti-CRBN antibodies, in certain embodiments, can also encompassantibodies which bind CRBN polypeptides and are encoded by nucleic acidsequences which are naturally occurring somatic variants of humangermline immunoglobulin nucleic acid sequence. In a specific embodiment,the anti-CRBN antibodies provided herein are fully human antibodies. Theterm “fully human antibody” includes antibodies having variable andconstant regions corresponding to human germline immunoglobulinsequences as described by Kabat et al., Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242, 1991. Exemplary methods ofproducing fully human antibodies are provided, e.g., in the Examplesherein, but any method known in the art may be used.

The phrase “recombinant human antibody” includes human antibodies thatare prepared, expressed, created or isolated by recombinant means, suchas antibodies expressed using a recombinant expression vectortransfected into a host cell, antibodies isolated from a recombinant,combinatorial human antibody library, antibodies isolated from an animal(e.g., a mouse or cow) that is transgenic and/or transchromosomal forhuman immunoglobulin genes (see, e.g., Taylor, L. D. et al. (1992) Nucl.Acids Res. 20:6287-6295) or antibodies prepared, expressed, created orisolated by any other means that involves splicing of humanimmunoglobulin gene sequences to other DNA sequences. Such recombinanthuman antibodies can have variable and constant regions derived fromhuman germline immunoglobulin sequences. See Kabat, E. A. et al. (1991)Sequences of Proteins of Immunological Interest, Fifth Edition, U.S.Department of Health and Human Services, NIH Publication No. 91-3242. Incertain embodiments, however, such recombinant human antibodies aresubjected to in vitro mutagenesis (or, when an animal transgenic forhuman Ig sequences is used, in vivo somatic mutagenesis) and thus theamino acid sequences of the VH and VL regions of the recombinantantibodies are sequences that, while derived from and related to humangermline VH and VL sequences, may not naturally exist within the humanantibody germline repertoire in vivo.

The term “heavy chain” when used in reference to an antibody refers tofive distinct types, called alpha (α), delta (δ), epsilon (ε), gamma (γ)and mu (μ), based on the amino acid sequence of the heavy chain constantdomain. These distinct types of heavy chains are well known and giverise to five classes of antibodies, IgA, IgD, IgE, IgG and IgM,respectively, including four subclasses of IgG, namely IgG1, IgG1, IgG3and IgG4. In some embodiments the heavy chain is a human heavy chain.

The terms “Kabat numbering,” and like terms are recognized in the artand refer to a system of numbering amino acid residues which are morevariable (i.e. hypervariable) than other amino acid residues in theheavy and light chain variable regions of an antibody, or an antigenbinding portion thereof. Kabat et al. (1971) Ann. any Acad. Sci.190:382-391 and, Kabat et al. (1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242. For the heavy chainvariable region, the hypervariable region typically ranges from aminoacid positions 31 to 35 for CDR1, amino acid positions 50 to 65 forCDR2, and amino acid positions 95 to 102 for CDR3. For the light chainvariable region, the hypervariable region typically ranges from aminoacid positions 24 to 34 for CDR1, amino acid positions 50 to 56 forCDR2, and amino acid positions 89 to 97 for CDR3. Other numberingschemes will be readily understood by those skilled in the art.

The term “light chain” when used in reference to an antibody refers totwo distinct types, called kappa (κ) of lambda (λ) based on the aminoacid sequence of the constant domains. Light chain amino acid sequencesare well known in the art. In certain embodiments, the light chain is ahuman light chain.

The term “monoclonal antibody” refers to an antibody obtained from apopulation of homogenous or substantially homogeneous antibodies, andeach monoclonal antibody will typically recognize a single epitope onthe antigen. In some embodiments, a “monoclonal antibody,” as usedherein, is an antibody produced by a single hybridoma or other cell,wherein the antibody immunospecifically binds to only a CRBN epitope asdetermined, e.g., by ELISA or other antigen-binding or competitivebinding assay known in the art or in the Examples provided herein. Theterm “monoclonal” is not limited to any particular method for making theantibody. For example, monoclonal antibodies provided herein may be madeby the hybridoma method as described in Kohler et al.; Nature, 256:495(1975) or may be isolated from phage libraries using the techniques asdescribed herein, for example. Other methods for the preparation ofclonal cell lines and of monoclonal antibodies expressed thereby arewell known in the art. See, e.g., Chapter 11 in: Short Protocols inMolecular Biology, (2002) 5th Ed., Ausubel et al., eds., John Wiley andSons, New York. Other exemplary methods of producing other monoclonalantibodies are provided in the Examples herein.

“Polyclonal antibodies” as used herein refers to an antibody populationgenerated in an immunogenic response to a protein having many epitopesand thus includes a variety of different antibodies directed to the sameand to different epitopes within the protein. Methods for producingpolyclonal antibodies are known in the art. See, e.g., Chapter 11 in:Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel et al.,eds., John Wiley and Sons, New York.

The terms “cereblon” or “CRBN” and similar terms refers to thepolypeptides (“polypeptides,” “peptides” and “proteins” are usedinterchangeably herein) comprising the amino acid sequence any CRBN,such as a human CRBN protein (e.g., human CRBN isoform 1, GenBankAccession No. NP_057386 (SEQ ID NO:12); or human CRBN isoforms 2,GenBank Accession No. NP_001166953 (SEQ ID NO:13), each of which isherein incorporated by reference in its entirety), and relatedpolypeptides, including SNP variants thereof. Related CRBN polypeptidesinclude allelic variants (e.g., SNP variants); splice variants;fragments; derivatives; substitution, deletion, and insertion variants;fusion polypeptides; and interspecies homologs, which, in certainembodiments, retain CRBN activity and/or are sufficient to generate ananti-CRBN immune response.

The term “CRBN antigen” refers to that portion of a CRBN polypeptide towhich an antibody immunospecifically binds. A CRBN antigen also refersto an analog or derivative of a CRBN polypeptide or fragment thereof towhich an antibody immunospecifically binds. A localized region on thesurface of a CRBN antigen that is capable of eliciting an immuneresponse is an CRBN “epitope.” A region of a CRBN polypeptidecontributing to an epitope may be contiguous amino acids of thepolypeptide or the epitope may come together from two or morenon-contiguous regions of the polypeptide. The epitope may or may not bea three-dimensional surface feature of the antigen. In certainembodiments, the CRBN epitope is EEFHGRTLHDDD (residues 1-12 of SEQ IDNO:1) or peptide 65-76 of human CRBN (SEQ ID NO:12).

The term “variable region” or “variable domain” refers to a portion ofthe light and heavy chains, typically about the amino-terminal 120 to130 amino acids in the heavy chain and about 100 to 110 amino acids inthe light chain, which differ extensively in sequence among antibodiesand are used in the binding and specificity of each particular antibodyfor its particular antigen. The variability in sequence is concentratedin those regions called complimentarily determining regions (CDRs) whilethe more highly conserved regions in the variable domain are calledframework regions (FR). The CDRs of the light and heavy chains areprimarily responsible for the interaction of the antibody with antigen.Numbering of amino acid positions used herein is according to the EUIndex, as in See Kabat, E. A. et al. (1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242. In some embodiments, thevariable region is a human variable region.

The term “expressed” or “expression” as used herein refers to thetranscription from a gene to give an RNA nucleic acid molecule at leastcomplementary in part to a region of one of the two nucleic acid strandsof the gene. The term “expressed” or “expression” as used herein alsorefers to the translation from the RNA molecule to give a protein, apolypeptide or a portion thereof.

An mRNA that is “upregulated” is generally increased upon a giventreatment or condition. An mRNA that is “downregulated” generally refersto a decrease in the level of expression of the mRNA in response to agiven treatment or condition. In some situations, the mRNA level canremain unchanged upon a given treatment or condition.

An mRNA from a patient sample can be “upregulated” when treated with adrug, as compared to a non-treated control. This upregulation can be,for example, an increase of about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,90%, 100%, 200%, 300%, 500%, 1,000%, 5,000% or more of the comparativecontrol mRNA level.

Alternatively, an mRNA can be “downregulated”, or expressed at a lowerlevel, in response to administration of certain compounds or otheragents. A downregulated mRNA can be, for example, present at a level ofabout 99%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 1% or lessof the comparative control mRNA level.

Similarly, the level of a polypeptide or protein biomarker from apatient sample can be increased when treated with a drug, as compared toa non-treated control. This increase can be about 5%, 10%, 20%, 30%,40%, 50%, 60%, 70%, 90%, 100%, 200%, 300%, 500%, 1,000%, 5,000% or moreof the comparative control protein level.

Alternatively, the level of a protein biomarker can be decreased inresponse to administration of certain compounds or other agents. Thisdecrease can be, for example, present at a level of about 99%, 95%, 90%,80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 1% or less of the comparativecontrol protein level.

The terms “determining”, “measuring”, “evaluating”, “assessing” and“assaying” as used herein generally refer to any form of measurement,and include determining if an element is present or not. These termsinclude both quantitative and/or qualitative determinations. Assessingmay be relative or absolute. “Assessing the presence of” can includedetermining the amount of something present, as well as determiningwhether it is present or absent.

The terms “nucleic acid” and “polynucleotide” are used interchangeablyherein to describe a polymer of any length composed of nucleotides,e.g., deoxyribonucleotides or ribonucleotides, or compounds producedsynthetically, which can hybridize with naturally occurring nucleicacids in a sequence specific manner analogous to that of two naturallyoccurring nucleic acids, e.g., can participate in Watson-Crick basepairing interactions. As used herein in the context of a polynucleotidesequence, the term “bases” (or “base”) is synonymous with “nucleotides”(or “nucleotide”), i.e., the monomer subunit of a polynucleotide. Theterms “nucleoside” and “nucleotide” are intended to include thosemoieties that contain not only the known purine and pyrimidine bases,but also other heterocyclic bases that have been modified. Suchmodifications include methylated purines or pyrimidines, acylatedpurines or pyrimidines, alkylated riboses or other heterocycles. Inaddition, the terms “nucleoside” and “nucleotide” include those moietiesthat contain not only conventional ribose and deoxyribose sugars, butother sugars as well. Modified nucleosides or nucleotides also includemodifications on the sugar moiety, e.g., wherein one or more of thehydroxyl groups are replaced with halogen atoms or aliphatic groups, orare functionalized as ethers, amines, or the like. “Analogues” refer tomolecules having structural features that are recognized in theliterature as being mimetics, derivatives, having analogous structures,or other like terms, and include, for example, polynucleotidesincorporating non-natural nucleotides, nucleotide mimetics such as2′-modified nucleosides, peptide nucleic acids, oligomeric nucleosidephosphonates, and any polynucleotide that has added substituent groups,such as protecting groups or linking moieties.

The term “complementary” refers to specific binding betweenpolynucleotides based on the sequences of the polynucleotides. As usedherein, a first polynucleotide and a second polynucleotide arecomplementary if they bind to each other in a hybridization assay understringent conditions, e.g. if they produce a given or detectable levelof signal in a hybridization assay. Portions of polynucleotides arecomplementary to each other if they follow conventional base-pairingrules, e.g. A pairs with T (or U) and G pairs with C, although smallregions (e.g. less than about 3 bases) of mismatch, insertion, ordeleted sequence may be present.

“Sequence identity” or “identity” in the context of two nucleic acidsequences refers to the residues in the two sequences which are the samewhen aligned for maximum correspondence over a specified comparisonwindow, and can take into consideration additions, deletions andsubstitutions.

The term “substantial identity” or “homologous” in their variousgrammatical forms in the context of polynucleotides generally means thata polynucleotide comprises a sequence that has a desired identity, forexample, at least 60% identity, preferably at least 70% sequenceidentity, more preferably at least 80%, still more preferably at least90% and even more preferably at least 95%, compared to a referencesequence. Another indication that nucleotide sequences are substantiallyidentical is if two molecules hybridize to each other under stringentconditions.

The terms “isolated” and “purified” refer to isolation of a substance(such as mRNA, antibody or protein) such that the substance comprises asubstantial portion of the sample in which it resides, i.e. greater thanthe substance is typically found in its natural or un-isolated state.Typically, a substantial portion of the sample comprises, e.g., greaterthan 1%, greater than 2%, greater than 5%, greater than 10%, greaterthan 20%, greater than 50%, or more, usually up to about 90%-100% of thesample. For example, a sample of isolated mRNA can typically comprise atleast about 1% total mRNA. Techniques for purifying polynucleotides arewell known in the art and include, for example, gel electrophoresis,ion-exchange chromatography, affinity chromatography, flow sorting, andsedimentation according to density.

The term “sample” as used herein relates to a material or mixture ofmaterials, typically, although not necessarily, in fluid form,containing one or more components of interest.

“Biological sample” as used herein refers to a sample obtained from abiological subject, including sample of biological tissue or fluidorigin, obtained, reached, or collected in vivo or in situ. A biologicalsample also includes samples from a region of a biological subjectcontaining precancerous or cancer cells or tissues. Such samples can be,but are not limited to, organs, tissues, fractions and cells isolatedfrom a mammal. Exemplary biological samples include but are not limitedto cell lysate, a cell culture, a cell line, a tissue, oral tissue,gastrointestinal tissue, an organ, an organelle, a biological fluid, ablood sample, a urine sample, a skin sample, and the like. Preferredbiological samples include but are not limited to whole blood, partiallypurified blood, PBMCs, tissue biopsies, and the like.

The term “capture agent,” as used herein, refers to an agent that bindsan mRNA or protein through an interaction that is sufficient to permitthe agent to bind and concentrate the mRNA or protein from a homogeneousmixture.

The term “probe” as used herein, refers to a capture agent that isdirected to a specific target mRNA biomarker sequence. Accordingly, eachprobe of a probe set has a respective target mRNA biomarker. Aprobe/target mRNA duplex is a structure formed by hybridizing a probe toits target mRNA biomarker.

The term “nucleic acid” or “oligonucleotide probe” refers to a nucleicacid capable of binding to a target nucleic acid of complementarysequence, such as the mRNA biomarkers provided herein, through one ormore types of chemical bonds, usually through complementary basepairing, usually through hydrogen bond formation. As used herein, aprobe may include natural (e.g., A, G, C, or T) or modified bases(7-deazaguanosine, inosine, etc.). In addition, the bases in a probe maybe joined by a linkage other than a phosphodiester bond, so long as itdoes not interfere with hybridization. It will be understood by one ofskill in the art that probes may bind target sequences lacking completecomplementarity with the probe sequence depending upon the stringency ofthe hybridization conditions. The probes are preferably directly labeledwith isotopes, for example, chromophores, lumiphores, chromogens, orindirectly labeled with biotin to which a streptavidin complex may laterbind. By assaying for the presence or absence of the probe, one candetect the presence or absence of a target mRNA biomarker of interest.

The term “stringent assay conditions” refers to conditions that arecompatible to produce binding pairs of nucleic acids, e.g., probes andtarget mRNAs, of sufficient complementarity to provide for the desiredlevel of specificity in the assay while being generally incompatible tothe formation of binding pairs between binding members of insufficientcomplementarity to provide for the desired specificity. The termstringent assay conditions generally refers to the combination ofhybridization and wash conditions.

A “label” or a “detectable moiety” in reference to a nucleic acid,refers to a composition that, when linked with a nucleic acid, rendersthe nucleic acid detectable, for example, by spectroscopic,photochemical, biochemical, immunochemical, or chemical means. Exemplarylabels include, but are not limited to, radioactive isotopes, magneticbeads, metallic beads, colloidal particles, fluorescent dyes, enzymes,biotin, digoxigenin, haptens, and the like. A “labeled nucleic acid oroligonucleotide probe” is generally one that is bound, eithercovalently, through a linker or a chemical bond, or noncovalently,through ionic bonds, van der Waals forces, electrostatic attractions,hydrophobic interactions, or hydrogen bonds, to a label such that thepresence of the nucleic acid or probe can be detected by detecting thepresence of the label bound to the nucleic acid or probe.

The terms “Polymerase chain reaction,” or “PCR,” as used hereingenerally refers to a procedure wherein small amounts of a nucleic acid,RNA and/or DNA, are amplified as described, for example, in U.S. Pat.No. 4,683,195 to Mullis. Generally, sequence information from the endsof the region of interest or beyond needs to be available, such thatoligonucleotide primers can be designed; these primers will be identicalor similar in sequence to opposite strands of the template to beamplified. The 5′ terminal nucleotides of the two primers may coincidewith the ends of the amplified material. PCR can be used to amplifyspecific RNA sequences, specific DNA sequences from total genomic DNA,and cDNA transcribed from total cellular RNA, bacteriophage or plasmidsequences, etc. See generally Mullis et al., Cold Spring Harbor Symp.Quant. Biol., 51: 263 (1987); Erlich, ed., PCR Technology, (StocktonPress, NY, 1989).

The term “cycle number” or “CT” when used herein in reference to PCRmethods, refers to the PCR cycle number at which the fluorescence levelpasses a given set threshold level. The CT measurement can be used, forexample, to approximate levels of mRNA in an original sample. The CTmeasurement is often used in terms of “dCT” or the “difference in theCT” score, when the CT of one nucleic acid is subtracted from the CT ofanother nucleic acid.

As used herein, and unless otherwise indicated, the term “opticallypure” means a composition that comprises one optical isomer of acompound and is substantially free of other isomers of that compound.For example, an optically pure composition of a compound having onechiral center will be substantially free of the opposite enantiomer ofthe compound. An optically pure composition of a compound having twochiral centers will be substantially free of other diastereomers of thecompound. A typical optically pure compound comprises greater than about80% by weight of one enantiomer of the compound and less than about 20%by weight of other enantiomers of the compound, more preferably greaterthan about 90% by weight of one enantiomer of the compound and less thanabout 10% by weight of the other enantiomers of the compound, even morepreferably greater than about 95% by weight of one enantiomer of thecompound and less than about 5% by weight of the other enantiomers ofthe compound, more preferably greater than about 97% by weight of oneenantiomer of the compound and less than about 3% by weight of the otherenantiomers of the compound, and most preferably greater than about 99%by weight of one enantiomer of the compound and less than about 1% byweight of the other enantiomers of the compound.

As used herein and unless otherwise indicated, the term“pharmaceutically acceptable salt” encompasses non-toxic acid and baseaddition salts of the compound to which the term refers. Acceptablenon-toxic acid addition salts include those derived from organic andinorganic acids or bases know in the art, which include, for example,hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid,methanesulphonic acid, acetic acid, tartaric acid, lactic acid, succinicacid, citric acid, malic acid, maleic acid, sorbic acid, aconitic acid,salicylic acid, phthalic acid, embolic acid, enanthic acid, and thelike.

Compounds that are acidic in nature are capable of forming salts withvarious pharmaceutically acceptable bases. The bases that can be used toprepare pharmaceutically acceptable base addition salts of such acidiccompounds are those that form non-toxic base addition salts, i.e., saltscontaining pharmacologically acceptable cations such as, but not limitedto, alkali metal or alkaline earth metal salts and the calcium,magnesium, sodium or potassium salts in particular. Suitable organicbases include, but are not limited to, N,N-dibenzylethylenediamine,chloroprocaine, choline, diethanolamine, ethylenediamine, meglumaine(N-methylglucamine), lysine, and procaine.

As used herein and unless otherwise indicated, the term “solvate” meansa compound provided herein or a salt thereof, that further includes astoichiometric or non-stoichiometric amount of solvent bound bynon-covalent intermolecular forces. Where the solvent is water, thesolvate is a hydrate.

As used herein and unless otherwise indicated, the term “stereomericallypure” means a composition that comprises one stereoisomer of a compoundand is substantially free of other stereoisomers of that compound. Forexample, a stereomerically pure composition of a compound having onechiral center will be substantially free of the opposite enantiomer ofthe compound. A stereomerically pure composition of a compound havingtwo chiral centers will be substantially free of other diastereomers ofthe compound. A typical stereomerically pure compound comprises greaterthan about 80% by weight of one stereoisomer of the compound and lessthan about 20% by weight of other stereoisomers of the compound, morepreferably greater than about 90% by weight of one stereoisomer of thecompound and less than about 10% by weight of the other stereoisomers ofthe compound, even more preferably greater than about 95% by weight ofone stereoisomer of the compound and less than about 5% by weight of theother stereoisomers of the compound, and most preferably greater thanabout 97% by weight of one stereoisomer of the compound and less thanabout 3% by weight of the other stereoisomers of the compound. As usedherein and unless otherwise indicated, the term “stereomericallyenriched” means a composition that comprises greater than about 60% byweight of one stereoisomer of a compound, preferably greater than about70% by weight, more preferably greater than about 80% by weight of onestereoisomer of a compound. As used herein and unless otherwiseindicated, the term “enantiomerically pure” means a stereomerically purecomposition of a compound having one chiral center. Similarly, the term“stereomerically enriched” means a stereomerically enriched compositionof a compound having one chiral center.

As used herein and unless otherwise indicated, the term “co-crystal”means a crystalline form that contains more than one compound in acrystal lattice. Co-crystals include crystalline molecular complexes oftwo or more non-volatile compounds bound together in a crystal latticethrough non-ionic interactions. As used herein, co-crystals includepharmaceutical cocrystals wherein the crystalline molecular complexescontainin a therapeutic compound and one or more additional non-volatilecompound(s) (referred to herein as counter-molecule(s)). Acounter-molecule in a pharmaceutical cocrystal is typically a non-toxicpharmaceutically acceptable molecule, such as, for example, foodadditives, preservatives, pharmaceutical excipients, or other APIs. Insome embodiments, pharmaceutical cocrystals enhance certainphysicochemical properties of drug products (e.g., solubility,dissolution rate, bioavailability and/or stability), withoutcompromising the chemical structural integrity of the activepharmaceutical ingredient (API). See, e.g., Jones et al.,“Pharmaceutical Cocrystals: An Emerging Approach to Physical PropertyEnhancement,” MRS Bulletin, 2006, 31, 875-879; Trask, “An Overview ofPharmaceutical Cocrystals as Intellectual Property,” MolecularPharmaceutics, 2007, 4(3), 301-309; Schultheiss & Newman,“Pharmaceutical Cocrystals and Their Physicochemical Properties,”Crystal Growth & Design, 2009, 9(6), 2950-2967; Shan & Zaworotko, “TheRole of Cocrystals in Pharmaceutical Science,” Drug Discovery Today,2008, 13(9/10), 440-446; and Vishweshwar et al., “PharmaceuticalCo-Crystals,” J. Pharm. Sci., 2006, 95(3), 499-516.

A biological marker or “biomarker” is a substance whose detectionindicates a particular biological state, such as, for example, thepresence of cancer. In some embodiments, biomarkers can either bedetermined individually, or several biomarkers can be measuredsimultaneously.

In some embodiments, a “biomarker” indicates a change in the level ofmRNA expression that may correlate with the risk or progression of adisease, or with the susceptibility of the disease to a given treatment.In some embodiments, the biomarker is a nucleic acid, such as a mRNA orcDNA.

In additional embodiments, a “biomarker” indicates a change in the levelof polypeptide or protein expression that may correlate with the risk,susceptibility to treatment, or progression of a disease. In someembodiments, the biomarker can be a polypeptide or protein, or afragment thereof. The relative level of specific proteins can bedetermined by methods known in the art. For example, antibody basedmethods, such as an immunoblot, enzyme-linked immunosorbent assay(ELISA), or other methods can be used.

It should be noted that if there is a discrepancy between a depictedstructure and a name given that structure, the depicted structure is tobe accorded more weight. In addition, if the stereochemistry of astructure or a portion of a structure is not indicated with, forexample, bold or dashed lines, the structure or portion of the structureis to be interpreted as encompassing all stereoisomers of it.

The practice of the embodiments provided herein will employ, unlessotherwise indicated, conventional techniques of molecular biology,microbiology, and immunology, which are within the skill of thoseworking in the art. Such techniques are explained fully in theliterature. Examples of particularly suitable texts for consultationinclude the following: Sambrook et al. (1989) Molecular Cloning; ALaboratory Manual (2d ed.); D. N Glover, ed. (1985) DNA Cloning, VolumesI and II; M. J. Gait, ed. (1984) Oligonucleotide Synthesis; B. D. Hames& S J. Higgins, eds. (1984) Nucleic Acid Hybridization; B. D. Hames & S.J. Higgins, eds. (1984) Transcription and Translation; R. I. Freshney,ed. (1986) Animal Cell Culture; Immobilized Cells and Enzymes (IRLPress, 1986); Immunochemical Methods in Cell and Molecular Biology(Academic Press, London); Scopes (1987) Protein Purification: Principlesand Practice (2d ed.; Springer Verlag, N.Y.); and D. M. Weir and C. C.Blackwell, eds. (1986) Handbook of Experimental Immunology, VolumesI-IV.

5.2 Clinical Trial Endpoints

“Overall survival” is defined as the time from randomization until deathfrom any cause, and is measured in the intent-to-treat population.Overall survival should be evaluated in randomized controlled studies.Demonstration of a statistically significant improvement in overallsurvival can be considered to be clinically significant if the toxicityprofile is acceptable, and has often supported new drug approval.

Several endpoints are based on cancer assessments. These endpointsinclude disease free survival (DFS), objective response rate (ORR), timeto progression (TTP), progression-free survival (PFS), andtime-to-treatment failure (TTF). The collection and analysis of data onthese time-dependent endpoints are based on indirect assessments,calculations, and estimates (e.g., tumor measurements).

Generally, “disease free survival” (DFS) is defined as the time fromrandomization until recurrence of cancer or death from any cause.Although overall survival is a conventional endpoint for most adjuvantsettings, DFS can be an important endpoint in situations where survivalmay be prolonged, making a survival endpoint impractical. DFS can be asurrogate for clinical benefit or it can provide direct evidence ofclinical benefit. This determination is based on the magnitude of theeffect, its risk-benefit relationship, and the disease setting. Thedefinition of DFS can be complicated, particularly when deaths are notedwithout prior cancer progression documentation. These events can bescored either as disease recurrences or as censored events. Although allmethods for statistical analysis of deaths have some limitations,considering all deaths (deaths from all causes) as recurrences canminimize bias. DFS can be overestimated using this definition,especially in patients who die after a long period without observation.Bias can be introduced if the frequency of long-term follow-up visits isdissimilar between the study arms or if dropouts are not random becauseof toxicity.

“Objective response rate” (ORR) is defined as the proportion of patientswith cancer reduction of a predefined amount and for a minimum timeperiod. Response duration usually is measured from the time of initialresponse until documented cancer progression. Generally, the FDA hasdefined ORR as the sum of partial responses plus complete responses.When defined in this manner, ORR is a direct measure of drug anticanceractivity, which can be evaluated in a single-arm study. If available,standardized criteria should be used to ascertain response. A variety ofresponse criteria have been considered appropriate (e.g., RECISTcriteria) (Therasse et al., (2000) J. Natl. Cancer Inst, 92: 205-16).The significance of ORR is assessed by its magnitude and duration, andthe percentage of complete responses (no detectable evidence of cancer).“Time to progression” (TTP) and “progression-free survival” (PFS) haveserved as primary endpoints for drug approval. TTP is defined as thetime from randomization until objective cancer progression; TTP does notinclude deaths. PFS is defined as the time from randomization untilobjective cancer progression or death. Compared with TTP, PFS is thepreferred regulatory endpoint. PFS includes deaths and thus can be abetter correlate to overall survival. PFS assumes patient deaths arerandomly related to cancer progression. However, in situations where themajority of deaths are unrelated to cancer, TTP can be an acceptableendpoint. As an endpoint to support drug approval, PFS can reflectcancer growth and be assessed before the determination of a survivalbenefit. Its determination is not confounded by subsequent therapy. Fora given sample size, the magnitude of effect on PFS can be larger thanthe effect on overall survival. However, the formal validation of PFS asa surrogate for survival for the many different malignancies that existcan be difficult. Data are sometimes insufficient to allow a robustevaluation of the correlation between effects on survival and PFS.Cancer trials are often small, and proven survival benefits of existingdrugs are generally modest. The role of PFS as an endpoint to supportlicensing approval varies in different cancer settings. Whether animprovement in PFS represents a direct clinical benefit or a surrogatefor clinical benefit depends on the magnitude of the effect and therisk-benefit of the new treatment compared to available therapies.

“Time-to-treatment failure” (TTF) is defined as a composite endpointmeasuring time from randomization to discontinuation of treatment forany reason, including disease progression, treatment toxicity, anddeath. TTF is not recommended as a regulatory endpoint for drugapproval. TTF does not adequately distinguish efficacy from theseadditional variables. A regulatory endpoint should clearly distinguishthe efficacy of the drug from toxicity, patient or physician withdrawal,or patient intolerance.

5.3 Second Active Agents

The compounds provided herein may be combined with otherpharmacologically active compounds (“second active agents”) in methodsand compositions provided herein. It is believed that certaincombinations work synergistically in the treatment of particular typesof cancer, and certain diseases and conditions associated with orcharacterized by undesired angiogenesis and/or inflammation. Thecompounds provided herein provided herein can also work to alleviateadverse effects associated with certain second active agents, and somesecond active agents can be used to alleviate adverse effects associatedwith the compounds provided herein provided herein.

One or more second active ingredients or agents can be used in themethods and compositions provided herein with the compounds providedherein. Second active agents can be large molecules (e.g., proteins) orsmall molecules (e.g., synthetic inorganic, organometallic, or organicmolecules).

Examples of large molecule active agents include, but are not limitedto, hematopoietic growth factors, cytokines, and monoclonal andpolyclonal antibodies. In certain embodiments, large molecule activeagents are biological molecules, such as naturally occurring orartificially made proteins. Proteins that are particularly useful inthis disclosure include proteins that stimulate the survival and/orproliferation of hematopoietic precursor cells and immunologicallyactive poietic cells in vitro or in vivo. Others stimulate the divisionand differentiation of committed erythroid progenitors in cells in vitroor in vivo. Particular proteins include, but are not limited to:interleukins, such as IL-2 (including recombinant IL-II (“rIL2”) andcanarypox IL-2), IL-10, IL-12, and IL-18; interferons, such asinterferon alfa-2a, interferon alfa-2b, interferon alfa-n1, interferonalfa-n3, interferon beta-I a, and interferon gamma-I b; GM-CF andGM-CSF; and EPO.

Particular proteins that can be used in the methods and compositions ofthe disclosure include, but are not limited to: filgrastim, which issold in the United States under the trade name NEUPOGEN® (Amgen,Thousand Oaks, Calif.); sargramostim, which is sold in the United Statesunder the trade name LEUKINE® (Immunex, Seattle, Wash.); and recombinantEPO, which is sold in the United States under the trade name EPGEN®(Amgen, Thousand Oaks, Calif.).

Recombinant and mutated forms of GM-CSF can be prepared as described inU.S. Pat. Nos. 5,391,485; 5,393,870; and 5,229,496; the disclosure ofeach of which is incorporated herein by reference in its entirety.Recombinant and mutated forms of G-CSF can be prepared as described inU.S. Pat. Nos. 4,810,643; 4,999,291; 5,528,823; and 5,580,755; thedisclosure of each of which is incorporated herein by reference in itsentirety.

This disclosure encompasses the use of native, naturally occurring, andrecombinant proteins. The disclosure further encompasses mutants andderivatives (e.g., modified forms) of naturally occurring proteins thatexhibit, in vivo, at least some of the pharmacological activity of theproteins upon which they are based. Examples of mutants include, but arenot limited to, proteins that have one or more amino acid residues thatdiffer from the corresponding residues in the naturally occurring formsof the proteins. Also encompassed by the term “mutants” are proteinsthat lack carbohydrate moieties normally present in their naturallyoccurring forms (e.g., nonglycosylated forms). Examples of derivativesinclude, but are not limited to, pegylated derivatives and fusionproteins, such as proteins formed by fusing IgG1 or IgG3 to the proteinor active portion of the protein of interest. See, e.g., Penichet, M. L.and Morrison, S. L., J. Immunol. Methods 248:91-101 (2001).

Antibodies that can be used in combination with the compound of FormulaI provided herein include monoclonal and polyclonal antibodies. Examplesof antibodies include, but are not limited to, trastuzumab (HERCEPTIN®),rituximab (RITUXAN®), bevacizumab (AVASTIN™), pertuzumab (OMNITARG™),tositumomab (BEXXAR®), edrecolomab (PANOREX®), panitumumab and G250. Thecompound of Formula I provided herein can also be combined with or usedin combination with anti-TNF-α antibodies.

Large molecule active agents may be administered in the form ofanti-cancer vaccines. For example, vaccines that secrete, or cause thesecretion of, cytokines such as IL-2, SCF, CXC14 (platelet factor 4),G-CSF, and GM-CSF can be used in the methods, pharmaceuticalcompositions, and kits of the disclosure. See, e.g., Emens, L. A., etal., Curr. Opinion Mol. Ther. 3(1):77-84 (2001).

Second active agents that are small molecules can also be used toalleviate adverse effects associated with the administration of thecompound of Formula I provided herein. However, like some largemolecules, many are believed to be capable of providing a synergisticeffect when administered with (e.g., before, after or simultaneously)the compound of Formula I. Examples of small molecule second activeagents include, but are not limited to, anti-cancer agents, antibiotics,immunosuppressive agents, and steroids.

Examples of anti-cancer agents include, but are not limited to:abraxane; ace-11; acivicin; aclarubicin; acodazole hydrochloride;acronine; adozelesin; aldesleukin; altretamine; ambomycin; ametantroneacetate; amrubicin; amsacrine; anastrozole; anthramycin; asparaginase;asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa;bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin;bleomycin sulfate; brequinar sodium; bropirimine; busulfan;cactinomycin; calusterone; caracemide; carbetimer; carboplatin;carmustine; carubicin hydrochloride; carzelesin; cedefingol; celecoxib(COX-2 inhibitor); chlorambucil; cirolemycin; cisplatin; cladribine;crisnatol mesylate; cyclophosphamide; cytarabine; dacarbazine;dactinomycin; daunorubicin hydrochloride; decitabine; dexormaplatin;dezaguanine; dezaguanine mesylate; diaziquone; docetaxel; doxorubicin;doxorubicin hydrochloride; droloxifene; droloxifene citrate;dromostanolone propionate; duazomycin; edatrexate; eflornithinehydrochloride; elsamitrucin; enloplatin; enpromate; epipropidine;epirubicin hydrochloride; erbulozole; esorubicin hydrochloride;estramustine; estramustine phosphate sodium; etanidazole; etoposide;etoposide phosphate; etoprine; fadrozole hydrochloride; fazarabine;fenretinide; floxuridine; fludarabine phosphate; fluorouracil;flurocitabine; fosquidone; fostriecin sodium; gemcitabine; gemcitabinehydrochloride; herceptin; hydroxyurea; idarubicin hydrochloride;ifosfamide; ilmofosine; iproplatin; irinotecan; irinotecanhydrochloride; lanreotide acetate; lapatinib; letrozole; leuprolideacetate; liarozole hydrochloride; lometrexol sodium; lomustine;losoxantrone hydrochloride; masoprocol; maytansine; mechlorethaminehydrochloride; megestrol acetate; melengestrol acetate; melphalan;menogaril; mercaptopurine; methotrexate; methotrexate sodium; metoprine;meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin; mitomalcin;mitomycin; mitosper; mitotane; mitoxantrone hydrochloride; mycophenolicacid; nocodazole; nogalamycin; ormaplatin; oxisuran; paclitaxel;pegaspargase; peliomycin; pentamustine; peplomycin sulfate;perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride;plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine;procarbazine hydrochloride; puromycin; puromycin hydrochloride;pyrazofurin; riboprine; romidepsin; safingol; safingol hydrochloride;semustine; simtrazene; sparfosate sodium; sparsomycin; spirogermaniumhydrochloride; spiromustine; spiroplatin; stem cell treatments such asPDA-001; streptonigrin; streptozocin; sulofenur; talisomycin; tecogalansodium; taxotere; tegafur; teloxantrone hydrochloride; temoporfin;teniposide; teroxirone; testolactone; thiamiprine; thioguanine;thiotepa; tiazofurin; tirapazamine; toremifene citrate; trestoloneacetate; triciribine phosphate; trimetrexate; trimetrexate glucuronate;triptorelin; tubulozole hydrochloride; uracil mustard; uredepa;vapreotide; verteporfin; vinblastine sulfate; vincristine sulfate;vindesine; vindesine sulfate; vinepidine sulfate; vinglycinate sulfate;vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate;vinzolidine sulfate; vorozole; zeniplatin; zinostatin; and zorubicinhydrochloride.

Other anti-cancer drugs include, but are not limited to: 20-epi-1,25dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin;acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TK antagonists;altretamine; ambamustine; amidox; amifostine; aminolevulinic acid;amrubicin; amsacrine; anagrelide; anastrozole; andrographolide;angiogenesis inhibitors; antagonist D; antagonist G; antarelix;anti-dorsalizing morphogenetic protein-1; antiandrogen, prostaticcarcinoma; antiestrogen; antineoplaston; antisense oligonucleotides;aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators;apurinic acid; ara-CDP-DL-PTBA; arginine deaminase; asulacrine;atamestane; atrimustine; axinastatin 1; axinastatin 2; axinastatin 3;azasetron; azatoxin; azatyrosine; baccatin III derivatives; balanol;batimastat; BCR/ABL antagonists; benzochlorins; benzoylstaurosporine;beta lactam derivatives; beta-alethine; betaclamycin B; betulinic acid;b-FGF inhibitor; bicalutamide; bisantrene; bisaziridinylspermine;bisnafide; bistratene A; bizelesin; breflate; bropirimine; budotitane;buthionine sulfoximine; calcipotriol; calphostin C; camptothecinderivatives; capecitabine; carboxamide-amino-triazole;carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor;carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropinB; cetrorelix; chlorins; chloroquinoxaline sulfonamide; cicaprost;cis-porphyrin; cladribine; clomifene analogues; clotrimazole;collismycin A; collismycin B; combretastatin A4; combretastatinanalogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8;cryptophycin A derivatives; curacin A; cyclopentanthraquinones;cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor;cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin;dexamethasone; dexifosfamide; dexrazoxane; dexverapamil; diaziquone;didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine;dihydrotaxol, 9-; dioxamycin; diphenyl spiromustine; docetaxel;docosanol; dolasetron; doxifluridine; doxorubicin; droloxifene;dronabinol; duocarmycin SA; ebselen; ecomustine; edelfosine;edrecolomab; eflornithine; elemene; emitefur; epirubicin; epristeride;estramustine analogue; estrogen agonists; estrogen antagonists;etanidazole; etoposide phosphate; exemestane; fadrozole; fazarabine;fenretinide; filgrastim; finasteride; flavopiridol; flezelastine;fluasterone; fludarabine; fluorodaunorunicin hydrochloride; forfenimex;formestane; fostriecin; fotemustine; gadolinium texaphyrin; galliumnitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine;glutathione inhibitors; hepsulfam; heregulin; hexamethylenebisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene;idramantone; ilmofosine; ilomastat; imatinib (e.g., GLEEVEC®),imiquimod; immunostimulant peptides; insulin-like growth factor-1receptor inhibitor; interferon agonists; interferons; interleukins;iobenguane; iododoxorubicin; ipomeanol, 4-; iroplact; irsogladine;isobengazole; isohomohalicondrin B; itasetron; jasplakinolide;kahalalide F; lamellarin-N triacetate; lanreotide; leinamycin;lenograstim; lentinan sulfate; leptolstatin; letrozole; leukemiainhibiting factor; leukocyte alpha interferon;leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole;linear polyamine analogue; lipophilic disaccharide peptide; lipophilicplatinum compounds; lissoclinamide 7; lobaplatin; lombricine;lometrexol; lonidamine; losoxantrone; loxoribine; lurtotecan; lutetiumtexaphyrin; lysofylline; lytic peptides; maitansine; mannostatin A;marimastat; masoprocol; maspin; matrilysin inhibitors; matrixmetalloproteinase inhibitors; menogaril; merbarone; meterelin;methioninase; metoclopramide; MIF inhibitor; mifepristone; miltefosine;mirimostim; mitoguazone; mitolactol; mitomycin analogues; mitonafide;mitotoxin fibroblast growth factor-saporin; mitoxantrone; mofarotene;molgramostim; Erbitux, human chorionic gonadotrophin; monophosphoryllipid A+myobacterium cell wall sk; mopidamol; mustard anticancer agent;mycaperoxide B; mycobacterial cell wall extract; myriaporone;N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip;naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin;nemorubicin; neridronic acid; nilutamide; nisamycin; nitric oxidemodulators; nitroxide antioxidant; nitrullyn; oblimersen (GENASENSE®);O⁶-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone;ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin;osaterone; oxaliplatin; oxaunomycin; paclitaxel; paclitaxel analogues;paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronic acid;panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase;peldesine; pentosan polysulfate sodium; pentostatin; pentrozole;perflubron; perfosfamide; perillyl alcohol; phenazinomycin;phenylacetate; phosphatase inhibitors; picibanil; pilocarpinehydrochloride; pirarubicin; piritrexim; placetin A; placetin B;plasminogen activator inhibitor; platinum complex; platinum compounds;platinum-triamine complex; porfimer sodium; porfiromycin; prednisone;propyl bis-acridone; prostaglandin J2; proteasome inhibitors; proteinA-based immune modulator; protein kinase C inhibitor; protein kinase Cinhibitors, microalgal; protein tyrosine phosphatase inhibitors; purinenucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine;pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists;raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors;ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re186 etidronate; rhizoxin; ribozymes; RII retinamide; rohitukine;romurtide; roquinimex; rubiginone B1; ruboxyl; safingol; saintopin;SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics; semustine;senescence derived inhibitor 1; sense oligonucleotides; signaltransduction inhibitors; sizofiran; sobuzoxane; sodium borocaptate;sodium phenylacetate; solverol; somatomedin binding protein; sonermin;sparfosic acid; spicamycin D; spiromustine; splenopentin; spongistatin1; squalamine; stipiamide; stromelysin inhibitors; sulfinosine;superactive vasoactive intestinal peptide antagonist; suradista;suramin; swainsonine; tallimustine; tamoxifen methiodide; tauromustine;tazarotene; tecogalan sodium; tegafur; tellurapyrylium; telomeraseinhibitors; temoporfin; teniposide; tetrachlorodecaoxide; tetrazomine;thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic;thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroidstimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocenebichloride; topsentin; toremifene; translation inhibitors; tretinoin;triacetyluridine; triciribine; trimetrexate; triptorelin; tropisetron;turosteride; tyrosine kinase inhibitors; tyrphostins; UBC inhibitors;ubenimex; urogenital sinus-derived growth inhibitory factor; urokinasereceptor antagonists; vapreotide; variolin B; velaresol; veramine;verdins; verteporfin; vinorelbine; vinxaltine; vitaxin; vorozole;zanoterone; zeniplatin; zilascorb; and zinostatin stimalamer.

Specific second active agents include, but are not limited to,oblimersen (GENASENSE®), remicade, docetaxel, celecoxib, melphalan,dexamethasone (DECADRON®), steroids, gemcitabine, cisplatinum,temozolomide, etoposide, cyclophosphamide, temodar, carboplatin,procarbazine, gliadel, tamoxifen, topotecan, methotrexate, ARISA®,taxol, taxotere, fluorouracil, leucovorin, irinotecan, xeloda, CPT-11,interferon alpha, pegylated interferon alpha (e.g., PEG INTRON-A),capecitabine, cisplatin, thiotepa, fludarabine, carboplatin, liposomaldaunorubicin, cytarabine, doxetaxol, pacilitaxel, vinblastine, IL-2,GM-CSF, dacarbazine, vinorelbine, zoledronic acid, palmitronate, biaxin,busulphan, prednisone, bisphosphonate, arsenic trioxide, vincristine,doxorubicin (DOXIL®), paclitaxel, ganciclovir, adriamycin, estramustinesodium phosphate (EMCYT®), sulindac, and etoposide.

5.4 Methods of Treatment and Prevention

In one embodiment, provided herein is a method of treating andpreventing cancer, which comprises administering to a patient a compoundprovided herein, or an enantiomer or a mixture of enantiomers thereof,or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal,clathrate, or polymorph thereof.

In another embodiment, provided herein is method of managing cancer,which comprises administering to a patient a compound provided herein,or an enantiomer or a mixture of enantiomers thereof, or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,clathrate, or polymorph thereof.

Also provided herein are methods of treating patients who have beenpreviously treated for cancer but are non-responsive to standardtherapies, as well as those who have not previously been treated. Theinvention also encompasses methods of treating patients regardless ofpatient's age, although some diseases or disorders are more common incertain age groups. The invention further encompasses methods oftreating patients who have undergone surgery in an attempt to treat thedisease or condition at issue, as well as those who have not. Becausepatients with cancer have heterogeneous clinical manifestations andvarying clinical outcomes, the treatment given to a patient may vary,depending on his/her prognosis. The skilled clinician will be able toreadily determine without undue experimentation specific secondaryagents, types of surgery, and types of non-drug based standard therapythat can be effectively used to treat an individual patient with cancer.

In yet another embodiment, provided herein is a method of treating,managing, or preventing diseases and disorders other than cancer thatare associated with or characterized by undesired angiogenesis, whichcomprises administering to a patient a compound provided herein, or anenantiomer or a mixture of enantiomers thereof, or a pharmaceuticallyacceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorphthereof.

As used herein, the term “cancer” includes, but is not limited to, solidtumors and blood born tumors. The term “cancer” refers to disease ofskin tissues, organs, blood, and vessels, including, but not limited to,cancers of the bladder, bone, blood, brain, breast, cervix, chest,colon, endrometrium, esophagus, eye, head, kidney, liver, lymph nodes,lung, mouth, neck, ovaries, pancreas, prostate, rectum, stomach, testis,throat, and uterus. Specific cancers include, but are not limited to,advanced malignancy, amyloidosis, neuroblastoma, meningioma,hemangiopericytoma, multiple brain metastase, glioblastoma multiforms,glioblastoma, brain stem glioma, poor prognosis malignant brain tumor,malignant glioma, recurrent malignant giolma, anaplastic astrocytoma,anaplastic oligodendroglioma, neuroendocrine tumor, rectaladenocarcinoma, Dukes C & D colorectal cancer, unresectable colorectalcarcinoma, metastatic hepatocellular carcinoma, Kaposi's sarcoma,karotype acute myeloblastic leukemia, Hodgkin's lymphoma, non-Hodgkin'slymphoma, cutaneous T-Cell lymphoma, cutaneous B-Cell lymphoma, diffuselarge B-Cell lymphoma, low grade follicular lymphoma, malignantmelanoma, malignant mesothelioma, malignant pleural effusionmesothelioma syndrome, peritoneal carcinoma, papillary serous carcinoma,gynecologic sarcoma, soft tissue sarcoma, scleroderma, cutaneousvasculitis, Langerhans cell histiocytosis, leiomyosarcoma,fibrodysplasia ossificans progressive, hormone refractory prostatecancer, resected high-risk soft tissue sarcoma, unrescectablehepatocellular carcinoma, Waldenstrom's macroglobulinemia, smolderingmyeloma, indolent myeloma, fallopian tube cancer, androgen independentprostate cancer, androgen dependent stage IV non-metastatic prostatecancer, hormone-insensitive prostate cancer, chemotherapy-insensitiveprostate cancer, papillary thyroid carcinoma, follicular thyroidcarcinoma, medullary thyroid carcinoma, and leiomyoma.

In certain embodiments, the cancer is a blood borne tumor. In certainembodiments, the blood borne tumor is metastatic. In certainembodiments, the blood borne tumor is drug resistant. In certainembodiments, the cancer is myeloma or lymphoma.

In certain embodiments, the cancer is a solid tumor. In certainembodiments, the solid tumor is metastatic. In certain embodiments, thesolid tumor is drug-resistant. In certain embodiments, the solid tumoris hepatocellular carcinoma, prostate cancer, ovarian cancer, orglioblastoma.

As used herein to refer to diseases and conditions other than cancer,the terms “diseases or disorders associated with or characterized byundesired angiogenesis,” “diseases or disorders associated withundesired angiogenesis,” and “diseases or disorders characterized byundesired angiogenesis” refer to diseases, disorders, and conditionsthat are caused, mediated, or attended by undesired, unwanted, oruncontrolled angiogenesis, including, but not limited to, inflammatorydiseases, autoimmune diseases, genetic diseases, allergic diseases,bacterial diseases, ocular neovascular diseases, choroidal neovasculardiseases, and retina neovascular diseases.

Examples of such diseases or disorders associated with undesiredangiogenesis include, but are not limited to, diabetic retinopathy,retinopathy of prematurity, corneal graft rejection, neovascularglaucoma, retrolental fibroplasia, proliferative vitreoretinopathy,trachoma, myopia, optic pits, epidemnic keratoconjunctivitis, atopickeratitis, superior limbic keratitis, pterygium keratitis sicca,sjogrens, acne rosacea, phylectenulosis, syphilis, lipid degeneration,bacterial ulcer, fungal ulcer, Herpes simplex infection, Herpes zosterinfection, protozoan infection, Kaposi sarcoma, Mooren ulcer, Terrien'smarginal degeneration, mariginal keratolysis, rheumatoid arthritis,systemic lupus, polyarteritis, trauma, Wegeners sarcoidosis, Scleritis,Steven's Johnson disease, periphigoid radial keratotomy, sickle cellanemia, sarcoid, pseudoxanthoma elasticum, Pagets disease, veinocclusion, artery occlusion, carotid obstructive disease, chronicuveitis, chronic vitritis, Lyme's disease, Eales disease, Behcet'sdisease, retinitis, choroiditis, presumed ocular histoplasmosis, Bestsdisease, Stargarts disease, pars planitis, chronic retinal detachment,hyperviscosity syndromes, toxoplasmosis, rubeosis, sarcodisis,sclerosis, soriatis, psoriasis, primary sclerosing cholangitis,proctitis, primary biliary srosis, idiopathic pulmonary fibrosis, andalcoholic hepatitis.

In certain embodiments, a therapeutically or prophylactically effectiveamount of the compound is from about 0.005 to about 1,000 mg per day,from about 0.01 to about 500 mg per day, from about 0.01 to about 250 mgper day, from about 0.01 to about 100 mg per day, from about 0.1 toabout 100 mg per day, from about 0.5 to about 100 mg per day, from about1 to about 100 mg per day, from about 0.01 to about 50 mg per day, fromabout 0.1 to about 50 mg per day, from about 0.5 to about 50 mg per day,from about 1 to about 50 mg per day, from about 0.02 to about 25 mg perday, or from about 0.05 to about 10 mg per day.

In certain embodiments, the therapeutically or prophylacticallyeffective amount is about 1, about 2, about 5, about 10, about 15, about20, about 25, about 30, about 40, about 45, about 50, about 60, about70, about 80, about 90, about 100, or about 150 mg per day.

In one embodiment, the recommended daily dose range of the compound forthe conditions described herein lie within the range of from about 0.5mg to about 50 mg per day, preferably given as a single once-a-day dose,or in divided doses throughout a day. In some embodiments, the dosageranges from about 1 mg to about 50 mg per day. In other embodiments, thedosage ranges from about 0.5 to about 5 mg per day. Specific doses perday include 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49 or 50 mg per day. In other embodiments, specific doses per dayinclude 0.5, 1, 1.5, 2, 2.5, 3, 3.5 or 4 mg per day. In a specificembodiment, the recommended starting dosage may be 0.5, 1, 1.5, 2, 2.5,3, 3.5, 4, 5, 10, 15, 20, 25 or 50 mg per day. In another embodiment,the recommended starting dosage may be 0.5, 1, 1.5, 2, 2.5, 3, 3.5 or 4mg per day. The dose may be escalated to 15, 20, 25, 30, 35, 40, 45 and50 mg/day. In a specific embodiment, the compound can be administered inan amount of about 25 mg/day to patients with cancer. In a particularembodiment, the compound can be administered in an amount of about 10mg/day to patients with cancer.

In certain embodiments, the therapeutically or prophylacticallyeffective amount is from about 0.001 to about 100 mg/kg/day, from about0.01 to about 50 mg/kg/day, from about 0.01 to about 25 mg/kg/day, fromabout 0.01 to about 10 mg/kg/day, from about 0.01 to about 9 mg/kg/day,0.01 to about 8 mg/kg/day, from about 0.01 to about 7 mg/kg/day, fromabout 0.01 to about 6 mg/kg/day, from about 0.01 to about 5 mg/kg/day,from about 0.01 to about 4 mg/kg/day, from about 0.01 to about 3mg/kg/day, from about 0.01 to about 2 mg/kg/day, or from about 0.01 toabout 1 mg/kg/day.

The administered dose can also be expressed in units other thanmg/kg/day. For example, doses for parenteral administration can beexpressed as mg/m²/day. One of ordinary skill in the art would readilyknow how to convert doses from mg/kg/day to mg/m²/day to given eitherthe height or weight of a subject or both (see,www.fda.gov/cder/cancer/animalframe.htm). For example, a dose of 1mg/kg/day for a 65 kg human is approximately equal to 38 mg/m²/day.

In certain embodiments, the amount of the compound administered issufficient to provide a plasma concentration of the compound at steadystate, ranging from about 0.001 to about 500 μM, about 0.002 to about200 μM, about 0.005 to about 100 μM, about 0.01 to about 50 μM, fromabout 1 to about 50 μM, about 0.02 to about 25 μM, from about 0.05 toabout 20 μM, from about 0.1 to about 20 μM, from about 0.5 to about 20μM, or from about 1 to about 20 μM.

In other embodiments, the amount of the compound administered issufficient to provide a plasma concentration of the compound at steadystate, ranging from about 5 to about 100 nM, about 5 to about 50 nM,about 10 to about 100 nM, about 10 to about 50 nM or from about 50 toabout 100 nM.

As used herein, the term “plasma concentration at steady state” is theconcentration reached after a period of administration of a compoundprovided herein, e.g., the compound of Formula I, or an enantiomer or amixture of enantiomers thereof, or a pharmaceutically acceptable salt,solvate, hydrate, co-crystal, clathrate, or polymorph thereof. Oncesteady state is reached, there are minor peaks and troughs on the timedependent curve of the plasma concentration of the compound.

In certain embodiments, the amount of the compound administered issufficient to provide a maximum plasma concentration (peakconcentration) of the compound, ranging from about 0.001 to about 500μM, about 0.002 to about 200 μM, about 0.005 to about 100 μM, about 0.01to about 50 μM, from about 1 to about 50 μM, about 0.02 to about 25 μM,from about 0.05 to about 20 μM, from about 0.1 to about 20 μM, fromabout 0.5 to about 20 μM, or from about 1 to about 20 μM.

In certain embodiments, the amount of the compound administered issufficient to provide a minimum plasma concentration (troughconcentration) of the compound, ranging from about 0.001 to about 500μM, about 0.002 to about 200 μM, about 0.005 to about 100 μM, about 0.01to about 50 μM, from about 1 to about 50 μM, about 0.01 to about 25 μM,from about 0.01 to about 20 μM, from about 0.02 to about 20 μM, fromabout 0.02 to about 20 μM, or from about 0.01 to about 20 μM.

In certain embodiments, the amount of the compound administered issufficient to provide an area under the curve (AUC) of the compound,ranging from about 100 to about 100,000 ng*hr/mL, from about 1,000 toabout 50,000 ng*hr/mL, from about 5,000 to about 25,000 ng*hr/mL, orfrom about 5,000 to about 10,000 ng*hr/mL.

In certain embodiments, the patient to be treated with one of themethods provided herein has not been treated with anticancer therapyprior to the administration of the drug. In certain embodiments, thepatient to be treated with one of the methods provided herein has beentreated with anticancer therapy prior to the administration of the drug.In certain embodiments, the patient to be treated with one of themethods provided herein has developed drug resistance to the anticancertherapy.

The methods provided herein encompass treating a patient regardless ofpatient's age, although some diseases or disorders are more common incertain age groups. Further provided herein is a method for treating apatient who has undergone surgery in an attempt to treat the disease orcondition at issue, as well in one who has not. Because the subjectswith cancer have heterogeneous clinical manifestations and varyingclinical outcomes, the treatment given to a particular subject may vary,depending on his/her prognosis. The skilled clinician will be able toreadily determine without undue experimentation, specific secondaryagents, types of surgery, and types of non-drug based standard therapythat can be effectively used to treat an individual subject with cancer.

Depending on the disease to be treated and the subject's condition, thecompound provided herein, or an enantiomer or a mixture of enantiomersthereof; or a pharmaceutically acceptable salt, solvate, hydrate,co-crystal, clathrate, or polymorph thereof, may be administered byoral, parenteral (e.g., intramuscular, intraperitoneal, intravenous,CIV, intracistemal injection or infusion, subcutaneous injection, orimplant), inhalation, nasal, vaginal, rectal, sublingual, or topical(e.g., transdermal or local) routes of administration. The compound, oran enantiomer or a mixture of enantiomers thereof; or a pharmaceuticallyacceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorphthereof, may be formulated, alone or together, in suitable dosage unitwith pharmaceutically acceptable excipients, carriers, adjuvants andvehicles, appropriate for each route of administration.

In one embodiment, the compound provided herein, or an enantiomer or amixture of enantiomers thereof; or a pharmaceutically acceptable salt,solvate, hydrate, co-crystal, clathrate, or polymorph thereof, isadministered orally. In another embodiment, the compound, or anenantiomer or a mixture of enantiomers thereof; or a pharmaceuticallyacceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorphthereof, is administered parenterally. In yet another embodiment, thecompound, or an enantiomer or a mixture of enantiomers thereof; or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,clathrate, or polymorph thereof, is administered intravenously.

The compound, or an enantiomer or a mixture of enantiomers thereof; or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,clathrate, or polymorph thereof, can be delivered as a single dose suchas, e.g., a single bolus injection, or oral tablets or pills; or overtime, such as, e.g., continuous infusion over time or divided bolusdoses over time. The compound can be administered repeatedly ifnecessary, for example, until the patient experiences stable disease orregression, or until the patient experiences disease progression orunacceptable toxicity. For example, stable disease for solid tumorsgenerally means that the perpendicular diameter of measurable lesionshas not increased by 25% or more from the last measurement. ResponseEvaluation Criteria in Solid Tumors (RECIST) Guidelines, Journal of theNational Cancer Institute 92(3): 205-216 (2000). Stable disease or lackthereof is determined by methods known in the art such as evaluation ofpatient symptoms, physical examination, visualization of the tumor thathas been imaged using X-ray, CAT, PET, or MM scan and other commonlyaccepted evaluation modalities.

The compound, or an enantiomer or a mixture of enantiomers thereof; or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,clathrate, or polymorph thereof, can be administered once daily (QD), ordivided into multiple daily doses such as twice daily (BID), three timesdaily (TID), and four times daily (QID). In addition, the administrationcan be continuous (i.e., daily for consecutive days or every day),intermittent, e.g., in cycles (i.e., including days, weeks, or months ofrest without drug). As used herein, the term “daily” is intended to meanthat a therapeutic compound is administered once or more than once eachday, for example, for a period of time. The term “continuous” isintended to mean that a therapeutic compound is administered daily foran uninterrupted period of at least 10 days to 52 weeks. The term“intermittent” or “intermittently” as used herein is intended to meanstopping and starting at either regular or irregular intervals. Forexample, intermittent administration of a compound is administration forone to six days per week, administration in cycles (e.g., dailyadministration for two to eight consecutive weeks, then a rest periodwith no administration for up to one week), or administration onalternate days. The term “cycling” as used herein is intended to meanthat a therapeutic compound is administered daily or continuously butwith a rest period.

In some embodiments, the frequency of administration is in the range ofabout a daily dose to about a monthly dose. In certain embodiments,administration is once a day, twice a day, three times a day, four timesa day, once every other day, twice a week, once every week, once everytwo weeks, once every three weeks, or once every four weeks. In oneembodiment, the compound, or an enantiomer or a mixture of enantiomersthereof; or a pharmaceutically acceptable salt, solvate, hydrate,co-crystal, clathrate, or polymorph thereof, is administered once a day.In another embodiment, the compound, or an enantiomer or a mixture ofenantiomers thereof; or a pharmaceutically acceptable salt, solvate,hydrate, co-crystal, clathrate, or polymorph thereof, is administeredtwice a day. In yet another embodiment, the compound, or an enantiomeror a mixture of enantiomers thereof; or a pharmaceutically acceptablesalt, solvate, hydrate, co-crystal, clathrate, or polymorph thereof, isadministered three times a day. In still another embodiment, thecompound, or an enantiomer or a mixture of enantiomers thereof; or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,clathrate, or polymorph thereof, is administered four times a day.

In certain embodiments, the compound, or an enantiomer or a mixture ofenantiomers thereof; or a pharmaceutically acceptable salt, solvate,hydrate, co-crystal, clathrate, or polymorph thereof, is administeredonce per day from one day to six months, from one week to three months,from one week to four weeks, from one week to three weeks, or from oneweek to two weeks. In certain embodiments, the compound, or apharmaceutically acceptable salt or solvate thereof, is administeredonce per day for one week, two weeks, three weeks, or four weeks. In oneembodiment, the compound, or an enantiomer or a mixture of enantiomersthereof; or a pharmaceutically acceptable salt, solvate, hydrate,co-crystal, clathrate, or polymorph thereof, is administered once perday for one week. In another embodiment, the compound, or an enantiomeror a mixture of enantiomers thereof; or a pharmaceutically acceptablesalt, solvate, hydrate, co-crystal, clathrate, or polymorph thereof, isadministered once per day for two weeks. In yet another embodiment, thecompound, or an enantiomer or a mixture of enantiomers thereof; or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,clathrate, or polymorph thereof, is administered once per day for threeweeks. In still another embodiment, the compound, or an enantiomer or amixture of enantiomers thereof; or a pharmaceutically acceptable salt,solvate, hydrate, co-crystal, clathrate, or polymorph thereof, isadministered once per day for four weeks.

5.5 Combination Therapy with a Second Active Agent

A compound provided herein, or an enantiomer or a mixture of enantiomersthereof; or a pharmaceutically acceptable salt, solvate, hydrate,co-crystal, clathrate, or polymorph thereof, can also be combined orused in combination with other therapeutic agents useful in thetreatment and/or prevention of a disease described herein.

In one embodiment, provided herein is a method of treating, preventing,or managing cancer, comprising administering to a patient compoundprovided herein, or an enantiomer or a mixture of enantiomers thereof;or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal,clathrate, or polymorph thereof; in combination with one or more secondactive agents, and optionally in combination with radiation therapy,blood transfusions, or surgery. Examples of second active agents aredisclosed herein (see, e.g., section 5.3).

As used herein, the term “in combination” includes the use of more thanone therapy (e.g., one or more prophylactic and/or therapeutic agents).However, the use of the term “in combination” does not restrict theorder in which therapies (e.g., prophylactic and/or therapeutic agents)are administered to a patient with a disease or disorder. A firsttherapy (e.g., a prophylactic or therapeutic agent such as a compoundprovided herein, a compound provided herein, or an enantiomer or amixture of enantiomers thereof, or a pharmaceutically acceptable salt,solvate, hydrate, co-crystal, clathrate, or polymorph thereof) can beadministered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequentto (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or12 weeks after) the administration of a second therapy (e.g., aprophylactic or therapeutic agent) to the subject. Triple therapy isalso contemplated herein.

Administration of the compound and one or more second active agents to apatient can occur simultaneously or sequentially by the same ordifferent routes of administration. The suitability of a particularroute of administration employed for a particular active agent willdepend on the active agent itself (e.g., whether it can be administeredorally without decomposing prior to entering the blood stream) and thecancer being treated.

The route of administration of the compound is independent of the routeof administration of a second therapy. In one embodiment, the compoundis administered orally. In another embodiment, the compound isadministered intravenously. Thus, in accordance with these embodiments,the compound is administered orally or intravenously, and the secondtherapy can be administered orally, parenterally, intraperitoneally,intravenously, intraarterially, transdermally, sublingually,intramuscularly, rectally, transbuccally, intranasally, liposomally, viainhalation, vaginally, intraoccularly, via local delivery by catheter orstent, subcutaneously, intraadiposally, intraarticularly, intrathecally,or in a slow release dosage form. In one embodiment, the compound and asecond therapy are administered by the same mode of administration,orally or by IV. In another embodiment, the compound is administered byone mode of administration, e.g., by IV, whereas the second agent (ananticancer agent) is administered by another mode of administration,e.g., orally.

In one embodiment, the second active agent is administered intravenouslyor subcutaneously and once or twice daily in an amount of from about 1to about 1000 mg, from about 5 to about 500 mg, from about 10 to about350 mg, or from about 50 to about 200 mg. The specific amount of thesecond active agent will depend on the specific agent used, the type ofdisease being treated or managed, the severity and stage of disease, andthe amount of the drug provided herein and any optional additionalactive agents concurrently administered to the patient. In certainembodiments, the second active agent is oblimersen (GENASENSE®), GM-CSF,G-CSF, SCF, EPO, taxotere, irinotecan, dacarbazine, transretinoic acid,topotecan, pentoxifylline, ciprofloxacin, dexamethasone, vincristine,doxorubicin, COX-2 inhibitor, IL2, IL8, IL18, IFN, Ara-C, vinorelbine,or a combination thereof.

In certain embodiments, GM-CSF, G-CSF, SCF or EPO is administeredsubcutaneously during about five days in a four or six week cycle in anamount ranging from about 1 to about 750 mg/m²/day, from about 25 toabout 500 mg/m²/day, from about 50 to about 250 mg/m²/day, or from about50 to about 200 mg/m²/day. In certain embodiments, GM-CSF may beadministered in an amount of from about 60 to about 500 mcg/m²intravenously over 2 hours or from about 5 to about 12 mcg/m²/daysubcutaneously. In certain embodiments, G-CSF may be administeredsubcutaneously in an amount of about 1 mcg/kg/day initially and can beadjusted depending on rise of total granulocyte counts. The maintenancedose of G-CSF may be administered in an amount of about 300 (in smallerpatients) or 480 mcg subcutaneously. In certain embodiments, EPO may beadministered subcutaneously in an amount of 10,000 Unit 3 times perweek.

In certain embodiments, a compound provided herein, or an enantiomer ora mixture of enantiomers thereof, or a pharmaceutically acceptable salt,solvate, hydrate, co-crystal, clathrate, or polymorph thereof, isadministered with melphalan and dexamethasone to patients withamyloidosis. In certain embodiments, a compound provided herein, e.g.,the compound of Formula I, or an enantiomer or a mixture of enantiomersthereof, or a pharmaceutically acceptable salt, solvate, hydrate,co-crystal, clathrate, or polymorph thereof, and steroids can beadministered to patients with amyloidosis.

In certain embodiments, a compound provided herein, or an enantiomer ora mixture of enantiomers thereof, or a pharmaceutically acceptable salt,solvate, hydrate, co-crystal, clathrate, or polymorph thereof, isadministered with gemcitabine and cisplatinum to patients with locallyadvanced or metastatic transitional cell bladder cancer.

In certain embodiments, a compound provided herein, or an enantiomer ora mixture of enantiomers thereof, or a pharmaceutically acceptable salt,solvate, hydrate, co-crystal, clathrate, or polymorph thereof, isadministered in combination with a second active ingredient as follows:temozolomide to pediatric patients with relapsed or progressive braintumors or recurrent neuroblastoma; celecoxib, etoposide andcyclophosphamide for relapsed or progressive CNS cancer; temodar topatients with recurrent or progressive meningioma, malignant meningioma,hemangiopericytoma, multiple brain metastases, relapased brain tumors,or newly diagnosed glioblastoma multiforms; irinotecan to patients withrecurrent glioblastoma; carboplatin to pediatric patients with brainstem glioma; procarbazine to pediatric patients with progressivemalignant gliomas; cyclophosphamide to patients with poor prognosismalignant brain tumors, newly diagnosed or recurrent glioblastomamultiforms; Gliadel® for high grade recurrent malignant gliomas;temozolomide and tamoxifen for anaplastic astrocytoma; or topotecan forgliomas, glioblastoma, anaplastic astrocytoma or anaplasticoligodendroglioma.

In certain embodiments, a compound provided herein, or an enantiomer ora mixture of enantiomers thereof, or a pharmaceutically acceptable salt,solvate, hydrate, co-crystal, clathrate, or polymorph thereof, isadministered with methotrexate, cyclophosphamide, taxane, abraxane,lapatinib, herceptin, aromatase inhibitors, selective estrogenmodulators, estrogen receptor antagonists, and/or PLX3397 (Plexxikon) topatients with metastatic breast cancer.

In certain embodiments, a compound provided herein, or an enantiomer ora mixture of enantiomers thereof, or a pharmaceutically acceptable salt,solvate, hydrate, co-crystal, clathrate, or polymorph thereof, isadministered with temozolomide to patients with neuroendocrine tumors.

In certain embodiments, a compound provided herein, or an enantiomer ora mixture of enantiomers thereof, or a pharmaceutically acceptable salt,solvate, hydrate, co-crystal, clathrate, or polymorph thereof, isadministered with gemcitabine to patients with recurrent or metastatichead or neck cancer.

In certain embodiments, a compound provided herein, or an enantiomer ora mixture of enantiomers thereof, or a pharmaceutically acceptable salt,solvate, hydrate, co-crystal, clathrate, or polymorph thereof, isadministered with gemcitabine to patients with pancreatic cancer.

In certain embodiments, a compound provided herein, or an enantiomer ora mixture of enantiomers thereof, or a pharmaceutically acceptable salt,solvate, hydrate, co-crystal, clathrate, or polymorph thereof, isadministered to patients with colon cancer in combination with ARISA®,avastatin, taxol, and/or taxotere.

In certain embodiments, a compound provided herein, or an enantiomer ora mixture of enantiomers thereof, or a pharmaceutically acceptable salt,solvate, hydrate, co-crystal, clathrate, or polymorph thereof, isadministered with capecitabine and/or PLX4032 (Plexxikon) to patientswith refractory colorectal cancer or patients who fail first linetherapy or have poor performance in colon or rectal adenocarcinoma.

In certain embodiments, a compound provided herein, or an enantiomer ora mixture of enantiomers thereof, or a pharmaceutically acceptable salt,solvate, hydrate, co-crystal, clathrate, or polymorph thereof, isadministered in combination with fluorouracil, leucovorin, andirinotecan to patients with Dukes C & D colorectal cancer or to patientswho have been previously treated for metastatic colorectal cancer.

In certain embodiments, a compound provided herein, or an enantiomer ora mixture of enantiomers thereof, or a pharmaceutically acceptable salt,solvate, hydrate, co-crystal, clathrate, or polymorph thereof, isadministered to patients with refractory colorectal cancer incombination with capecitabine, xeloda, and/or CPT-11.

In certain embodiments, a compound provided herein, or an enantiomer ora mixture of enantiomers thereof, or a pharmaceutically acceptable salt,solvate, hydrate, co-crystal, clathrate, or polymorph thereof, isadministered with capecitabine and irinotecan to patients withrefractory colorectal cancer or to patients with unresectable ormetastatic colorectal carcinoma.

In certain embodiments, a compound provided herein, or an enantiomer ora mixture of enantiomers thereof, or a pharmaceutically acceptable salt,solvate, hydrate, co-crystal, clathrate, or polymorph thereof, isadministered alone or in combination with interferon alpha orcapecitabine to patients with unresectable or metastatic hepatocellularcarcinoma; or with cisplatin and thiotepa to patients with primary ormetastatic liver cancer.

In certain embodiments, a compound provided herein, or an enantiomer ora mixture of enantiomers thereof, or a pharmaceutically acceptable salt,solvate, hydrate, co-crystal, clathrate, or polymorph thereof, isadministered in combination with pegylated interferon alpha to patientswith Kaposi's sarcoma.

In certain embodiments, a compound provided herein, or an enantiomer ora mixture of enantiomers thereof, or a pharmaceutically acceptable salt,solvate, hydrate, co-crystal, clathrate, or polymorph thereof, isadministered in combination with fludarabine, carboplatin, and/ortopotecan to patients with refractory or relapsed or high-risk acutedmyelogenous leukemia.

In certain embodiments, a compound provided herein, or an enantiomer ora mixture of enantiomers thereof, or a pharmaceutically acceptable salt,solvate, hydrate, co-crystal, clathrate, or polymorph thereof, isadministered in combination with liposomal daunorubicin, topotecanand/or cytarabine to patients with unfavorable karotype acutemyeloblastic leukemia.

In certain embodiments, a compound provided herein, or an enantiomer ora mixture of enantiomers thereof, or a pharmaceutically acceptable salt,solvate, hydrate, co-crystal, clathrate, or polymorph thereof, isadministered in combination with gemcitabine, abraxane, erlotinib,geftinib, and/or irinotecan to patients with non-small cell lung cancer.

In certain embodiments, a compound provided herein, or an enantiomer ora mixture of enantiomers thereof, or a pharmaceutically acceptable salt,solvate, hydrate, co-crystal, clathrate, or polymorph thereof, isadministered in combination with carboplatin and irinotecan to patientswith non-small cell lung cancer.

In certain embodiments, a compound provided herein, or an enantiomer ora mixture of enantiomers thereof, or a pharmaceutically acceptable salt,solvate, hydrate, co-crystal, clathrate, or polymorph thereof, isadministered with doxetaxol to patients with non-small cell lung cancerwho have been previously treated with carbo/VP 16 and radiotherapy.

In certain embodiments, a compound provided herein, or an enantiomer ora mixture of enantiomers thereof, or a pharmaceutically acceptable salt,solvate, hydrate, co-crystal, clathrate, or polymorph thereof, isadministered in combination with carboplatin and/or taxotere, or incombination with carboplatin, pacilitaxel and/or thoracic radiotherapyto patients with non-small cell lung cancer.

In certain embodiments, a compound provided herein, or an enantiomer ora mixture of enantiomers thereof, or a pharmaceutically acceptable salt,solvate, hydrate, co-crystal, clathrate, or polymorph thereof, isadministered in combination with taxotere to patients with stage IIIB orIV non-small cell lung cancer.

In certain embodiments, a compound provided herein, or an enantiomer ora mixture of enantiomers thereof, or a pharmaceutically acceptable salt,solvate, hydrate, co-crystal, clathrate, or polymorph thereof, isadministered in combination with oblimersen (Genasense®) to patientswith small cell lung cancer.

In certain embodiments, a compound provided herein, e.g., the compoundof Formula I, or an enantiomer or a mixture of enantiomers thereof, or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,clathrate, or polymorph thereof, is administered in combination withABT-737 (Abbott Laboratories) and/or obatoclax (GX15-070) to patientswith lymphoma and other blood cancers.

In certain embodiments, a compound provided herein, or an enantiomer ora mixture of enantiomers thereof, or a pharmaceutically acceptable salt,solvate, hydrate, co-crystal, clathrate, or polymorph thereof, isadministered alone or in combination with a second active ingredientsuch as vinblastine or fludarabine to patients with various types oflymphoma, including, but not limited to, Hodgkin's lymphoma,non-Hodgkin's lymphoma, cutaneous T-Cell lymphoma, cutaneous B-Celllymphoma, diffuse large B-Cell lymphoma or relapsed or refractory lowgrade follicular lymphoma.

In certain embodiments, a compound provided herein, or an enantiomer ora mixture of enantiomers thereof, or a pharmaceutically acceptable salt,solvate, hydrate, co-crystal, clathrate, or polymorph thereof, isadministered in combination with taxotere, IL-2, IFN, GM-CSF, PLX4032(Plexxikon) and/or dacarbazine to patients with various types or stagesof melanoma.

In certain embodiments, a compound provided herein, or an enantiomer ora mixture of enantiomers thereof, or a pharmaceutically acceptable salt,solvate, hydrate, co-crystal, clathrate, or polymorph thereof, isadministered alone or in combination with vinorelbine to patients withmalignant mesothelioma, or stage IIIB non-small cell lung cancer withpleural implants or malignant pleural effusion mesothelioma syndrome.

In certain embodiments, a compound provided herein, or an enantiomer ora mixture of enantiomers thereof, or a pharmaceutically acceptable salt,solvate, hydrate, co-crystal, clathrate, or polymorph thereof, isadministered to patients with various types or stages of multiplemyeloma in combination with dexamethasone, zoledronic acid,palmitronate, GM-CSF, biaxin, vinblastine, melphalan, busulphan,cyclophosphamide, IFN, palmidronate, prednisone, bisphosphonate,celecoxib, arsenic trioxide, PEG INTRON-A, vincristine, or a combinationthereof.

In certain embodiments, a compound provided herein, or an enantiomer ora mixture of enantiomers thereof, or a pharmaceutically acceptable salt,solvate, hydrate, co-crystal, clathrate, or polymorph thereof, isadministered to patients with relapsed or refractory multiple myeloma incombination with doxorubicin (Doxil®), vincristine and/or dexamethasone(Decadron®).

In certain embodiments, a compound provided herein, or an enantiomer ora mixture of enantiomers thereof, or a pharmaceutically acceptable salt,solvate, hydrate, co-crystal, clathrate, or polymorph thereof, isadministered to patients with various types or stages of ovarian cancersuch as peritoneal carcinoma, papillary serous carcinoma, refractoryovarian cancer or recurrent ovarian cancer, in combination with taxol,carboplatin, doxorubicin, gemcitabine, cisplatin, xeloda, paclitaxel,dexamethasone, or a combination thereof.

In certain embodiments, a compound provided herein, or an enantiomer ora mixture of enantiomers thereof, or a pharmaceutically acceptable salt,solvate, hydrate, co-crystal, clathrate, or polymorph thereof, isadministered to patients with various types or stages of prostatecancer, in combination with xeloda, 5 FU/LV, gemcitabine, irinotecanplus gemcitabine, cyclophosphamide, vincristine, dexamethasone, GM-CSF,celecoxib, taxotere, ganciclovir, paclitaxel, adriamycin, docetaxel,estramustine, Emcyt, denderon or a combination thereof.

In certain embodiments, a compound provided herein, or an enantiomer ora mixture of enantiomers thereof, or a pharmaceutically acceptable salt,solvate, hydrate, co-crystal, clathrate, or polymorph thereof, isadministered to patients with various types or stages of renal cellcancer, in combination with capecitabine, IFN, tamoxifen, IL-2, GM-CSF,Celebrex®, or a combination thereof.

In certain embodiments, a compound provided herein, or an enantiomer ora mixture of enantiomers thereof, or a pharmaceutically acceptable salt,solvate, hydrate, co-crystal, clathrate, or polymorph thereof, isadministered to patients with various types or stages of gynecologic,uterus or soft tissue sarcoma cancer in combination with IFN, a COX-2inhibitor such as Celebrex®, and/or sulindac.

In certain embodiments, a compound provided herein, or an enantiomer ora mixture of enantiomers thereof, or a pharmaceutically acceptable salt,solvate, hydrate, co-crystal, clathrate, or polymorph thereof, isadministered to patients with various types or stages of solid tumors incombination with celebrex, etoposide, cyclophosphamide, docetaxel,apecitabine, IFN, tamoxifen, IL-2, GM-CSF, or a combination thereof.

In certain embodiments, a compound provided herein, or an enantiomer ora mixture of enantiomers thereof, or a pharmaceutically acceptable salt,solvate, hydrate, co-crystal, clathrate, or polymorph thereof, isadministered to patients with scleroderma or cutaneous vasculitis incombination with celebrex, etoposide, cyclophosphamide, docetaxel,apecitabine, IFN, tamoxifen, IL-2, GM-CSF, or a combination thereof.

Also encompassed herein is a method of increasing the dosage of ananti-cancer drug or agent that can be safely and effectivelyadministered to a patient, which comprises administering to the patient(e.g., a human) or an enantiomer or a mixture of enantiomers thereof, ora pharmaceutically acceptable salt, solvate, hydrate, co-crystal,clathrate, or polymorph thereof. Patients that can benefit by thismethod are those likely to suffer from an adverse effect associated withanti-cancer drugs for treating a specific cancer of the skin,subcutaneous tissue, lymph nodes, brain, lung, liver, bone, intestine,colon, heart, pancreas, adrenal, kidney, prostate, breast, colorectal,or combinations thereof. The administration of a compound providedherein, or an enantiomer or a mixture of enantiomers thereof, or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,clathrate, or polymorph thereof, alleviates or reduces adverse effectswhich are of such severity that it would otherwise limit the amount ofanti-cancer drug.

In one embodiment, a compound provided herein, or an enantiomer or amixture of enantiomers thereof, or a pharmaceutically acceptable salt,solvate, hydrate, co-crystal, clathrate, or polymorph thereof, isadministered orally and daily in an amount ranging from about 0.1 toabout 150 mg, from about 1 to about 50 mg, or from about 2 to about 25mg, prior to, during, or after the occurrence of the adverse effectassociated with the administration of an anti-cancer drug to a patient.In certain embodiments, a compound provided herein, or an enantiomer ora mixture of enantiomers thereof, or a pharmaceutically acceptable salt,solvate, hydrate, co-crystal, clathrate, or polymorph thereof, isadministered in combination with specific agents such as heparin,aspirin, coumadin, or G-CSF to avoid adverse effects that are associatedwith anti-cancer drugs such as but not limited to neutropenia orthrombocytopenia.

In one embodiment, a compound provided herein, or an enantiomer or amixture of enantiomers thereof, or a pharmaceutically acceptable salt,solvate, hydrate, co-crystal, clathrate, or polymorph thereof, isadministered to patients with diseases and disorders associated with orcharacterized by, undesired angiogenesis in combination with additionalactive ingredients, including, but not limited to, anti-cancer drugs,anti-inflammatories, antihistamines, antibiotics, and steroids.

In another embodiment, encompassed herein is a method of treating,preventing and/or managing cancer, which comprises administering thecompound, or an enantiomer or a mixture of enantiomers thereof, or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,clathrate, or polymorph thereof, in conjunction with (e.g. before,during, or after) conventional therapy including, but not limited to,surgery, immunotherapy, biological therapy, radiation therapy, or othernon-drug based therapy presently used to treat, prevent or managecancer. The combined use of the compound provided herein andconventional therapy may provide a unique treatment regimen that isunexpectedly effective in certain patients. Without being limited bytheory, it is believed that the compound of Formula I may provideadditive or synergistic effects when given concurrently withconventional therapy.

As discussed elsewhere herein, encompassed herein is a method ofreducing, treating and/or preventing adverse or undesired effectsassociated with conventional therapy including, but not limited to,surgery, chemotherapy, radiation therapy, hormonal therapy, biologicaltherapy and immunotherapy. A compound provided herein, or an enantiomeror a mixture of enantiomers thereof, or a pharmaceutically acceptablesalt, solvate, hydrate, co-crystal, clathrate, or polymorph thereof, andother active ingredient can be administered to a patient prior to,during, or after the occurrence of the adverse effect associated withconventional therapy.

In one embodiment, the compound can be administered in an amount rangingfrom about 0.1 to about 150 mg, from about 1 to about 25 mg, from about2 to about 10 mg, or about 0.5 to about 4 mg orally and daily alone, orin combination with a second active agent disclosed herein (see, e.g.,section 4.3), prior to, during, or after the use of conventionaltherapy.

In certain embodiments, a compound provided herein, or an enantiomer ora mixture of enantiomers thereof, or a pharmaceutically acceptable salt,solvate, hydrate, co-crystal, clathrate, or polymorph thereof, anddoxetaxol are administered to patients with non-small cell lung cancerwho were previously treated with carbo/VP 16 and radiotherapy.

5.6 Pharmaceutical Compositions

Pharmaceutical compositions can be used in the preparation ofindividual, single unit dosage forms. Pharmaceutical compositions anddosage forms provided herein comprise a compound, or a pharmaceuticallyacceptable salt, solvate, hydrate, stereoisomer, clathrate, or prodrugthereof. Pharmaceutical compositions and dosage forms provided hereinmay further comprise one or more excipients.

Pharmaceutical compositions and dosage forms provided herein may alsocomprise one or more additional active ingredients. Examples of optionalsecond, or additional, active ingredients are disclosed herein.

Single unit dosage forms are suitable for oral, mucosal (e.g., nasal,sublingual, vaginal, buccal, or rectal), parenteral (e.g., subcutaneous,intravenous, bolus injection, intramuscular, or intraarterial), topical(e.g., eye drops or other ophthalmic preparations), transdermal ortranscutaneous administration to a patient. Examples of dosage formsinclude, but are not limited to: tablets; caplets; capsules, such assoft elastic gelatin capsules; cachets; troches; lozenges; dispersions;suppositories; powders; aerosols (e.g., nasal sprays or inhalers); gels;liquid dosage forms suitable for oral or mucosal administration to apatient, including suspensions (e.g., aqueous or non-aqueous liquidsuspensions, oil-in-water emulsions, or a water-in-oil liquidemulsions), solutions, and elixirs; liquid dosage forms suitable forparenteral administration to a patient; eye drops or other ophthalmicpreparations suitable for topical administration; and sterile solids(e.g., crystalline or amorphous solids) that can be reconstituted toprovide liquid dosage forms suitable for parenteral administration to apatient.

The composition, shape, and type of dosage forms provided herein willtypically vary depending on their use. For example, a dosage form usedin the acute treatment of a disease may contain larger amounts of one ormore of the active ingredients it comprises than a dosage form used inthe chronic treatment of the same disease. Similarly, a parenteraldosage form may contain smaller amounts of one or more of the activeingredients it comprises than an oral dosage form used to treat the samedisease. These and other ways in which specific dosage forms providedherein will vary from one another will be readily apparent to thoseskilled in the art. See, e.g., Remington's Pharmaceutical Sciences, 18thed., Mack Publishing, Easton Pa. (1990).

Typical pharmaceutical compositions and dosage forms comprise one ormore excipients. Suitable excipients are well known to those skilled inthe art of pharmacy, and non-limiting examples of suitable excipientsare provided herein. Whether a particular excipient is suitable forincorporation into a pharmaceutical composition or dosage form dependson a variety of factors well known in the art including, but not limitedto, the way in which the dosage form will be administered to a patient.For example, oral dosage forms such as tablets may contain excipientsnot suited for use in parenteral dosage forms. The suitability of aparticular excipient may also depend on the specific active ingredientsin the dosage form. For example, the decomposition of some activeingredients may be accelerated by some excipients such as lactose, orwhen exposed to water. Active ingredients that comprise primary orsecondary amines are particularly susceptible to such accelerateddecomposition. Consequently, provided herein are pharmaceuticalcompositions and dosage forms that contain little, if any, lactose othermono- or di-saccharides. As used herein, the term “lactose-free” meansthat the amount of lactose present, if any, is insufficient tosubstantially increase the degradation rate of an active ingredient.

Lactose-free compositions provided herein can comprise excipients thatare well known in the art and are listed, for example, in the U.S.Pharmacopeia (USP) 25-NF20 (2002). In general, lactose-free compositionscomprise active ingredients, a binder/filler, and a lubricant inpharmaceutically compatible and pharmaceutically acceptable amounts. Inone embodiment, lactose-free dosage forms comprise active ingredients,microcrystalline cellulose, pre-gelatinized starch, and magnesiumstearate.

Also provided herein are anhydrous pharmaceutical compositions anddosage forms comprising active ingredients, since water can facilitatethe degradation of some compounds. For example, the addition of water(e.g., 5%) is widely accepted in the pharmaceutical arts as a means ofsimulating long-term storage in order to determine characteristics suchas shelf-life or the stability of formulations over time. See, e.g.,Jens T. Carstensen, Drug Stability: Principles & Practice, 2d. Ed.,Marcel Dekker, NY, N.Y., 1995, pp. 379-80. In effect, water and heataccelerate the decomposition of some compounds. Thus, the effect ofwater on a formulation can be of great significance since moistureand/or humidity are commonly encountered during manufacture, handling,packaging, storage, shipment, and use of formulations.

Anhydrous pharmaceutical compositions and dosage forms may be preparedusing anhydrous or low moisture containing ingredients and low moistureor low humidity conditions. Pharmaceutical compositions and dosage formsthat comprise lactose and at least one active ingredient that comprisesa primary or secondary amine are preferably anhydrous if substantialcontact with moisture and/or humidity during manufacturing, packaging,and/or storage is expected.

An anhydrous pharmaceutical composition should be prepared and storedsuch that its anhydrous nature is maintained. Accordingly, anhydrouscompositions are preferably packaged using materials known to preventexposure to water such that they can be included in suitable formularykits. Examples of suitable packaging include, but are not limited to,hermetically sealed foils, plastics, unit dose containers (e.g., vials),blister packs, and strip packs.

Also provided herein are pharmaceutical compositions and dosage formsthat comprise one or more compounds that reduce the rate by which anactive ingredient will decompose. Such compounds, which are referred toherein as “stabilizers,” include, but are not limited to, antioxidantssuch as ascorbic acid, pH buffers, or salt buffers.

5.7 Oral Dosage Forms

Pharmaceutical compositions that are suitable for oral administrationcan be presented as discrete dosage forms, such as, but are not limitedto, tablets (e.g., chewable tablets), caplets, capsules, and liquids(e.g., flavored syrups). Such dosage forms contain predetermined amountsof active ingredients, and may be prepared by methods of pharmacy wellknown to those skilled in the art. See generally, Remington'sPharmaceutical Sciences, 18th ed., Mack Publishing, Easton Pa. (1990).

Typical oral dosage forms are prepared by combining the activeingredients in an intimate admixture with at least one excipientaccording to conventional pharmaceutical compounding techniques.Excipients can take a wide variety of forms depending on the form ofpreparation desired for administration. For example, excipients suitablefor use in oral liquid or aerosol dosage forms include, but are notlimited to, water, glycols, oils, alcohols, flavoring agents,preservatives, and coloring agents. Examples of excipients suitable foruse in solid oral dosage forms (e.g., powders, tablets, capsules, andcaplets) include, but are not limited to, starches, sugars,micro-crystalline cellulose, diluents, granulating agents, lubricants,binders, and disintegrating agents.

Because of their ease of administration, tablets and capsules representthe most advantageous oral dosage unit forms, in which case solidexcipients are employed. If desired, tablets can be coated by standardaqueous or nonaqueous techniques. Such dosage forms can be prepared byany of the methods of pharmacy. In general, pharmaceutical compositionsand dosage forms are prepared by uniformly and intimately admixing theactive ingredients with liquid carriers, finely divided solid carriers,or both, and then shaping the product into the desired presentation ifnecessary.

For example, a tablet can be prepared by compression or molding.Compressed tablets can be prepared by compressing in a suitable machinethe active ingredients in a free-flowing form such as powder orgranules, optionally mixed with an excipient. Molded tablets can be madeby molding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

Examples of excipients that can be used in oral dosage forms providedherein include, but are not limited to, binders, fillers, disintegrants,and lubricants. Binders suitable for use in pharmaceutical compositionsand dosage forms include, but are not limited to, corn starch, potatostarch, or other starches, gelatin, natural and synthetic gums such asacacia, sodium alginate, alginic acid, other alginates, powderedtragacanth, guar gum, cellulose and its derivatives (e.g., ethylcellulose, cellulose acetate, carboxymethyl cellulose calcium, sodiumcarboxymethyl cellulose), polyvinyl pyrrolidone, methyl cellulose,pre-gelatinized starch, hydroxypropyl methyl cellulose, (e.g., Nos.2208, 2906, 2910), microcrystalline cellulose, and mixtures thereof.

Suitable forms of microcrystalline cellulose include, but are notlimited to, the materials sold as AVICEL-PH-101, AVICEL-PH-103 AVICELRC-581, AVICEL-PH-105 (available from FMC Corporation, American ViscoseDivision, Avicel Sales, Marcus Hook, Pa.), and mixtures thereof. Anspecific binder is a mixture of microcrystalline cellulose and sodiumcarboxymethyl cellulose sold as AVICEL RC-581. Suitable anhydrous or lowmoisture excipients or additives include AVICEL-PH-103™ and Starch 1500LM.

Examples of fillers suitable for use in the pharmaceutical compositionsand dosage forms disclosed herein include, but are not limited to, talc,calcium carbonate (e.g., granules or powder), microcrystallinecellulose, powdered cellulose, dextrates, kaolin, mannitol, silicicacid, sorbitol, starch, pre-gelatinized starch, and mixtures thereof.The binder or filler in pharmaceutical compositions of the invention istypically present in from about 50 to about 99 weight percent of thepharmaceutical composition or dosage form.

Disintegrants are used in compositions to provide tablets thatdisintegrate when exposed to an aqueous environment. Tablets thatcontain too much disintegrant may disintegrate in storage, while thosethat contain too little may not disintegrate at a desired rate or underthe desired conditions. Thus, a sufficient amount of disintegrant thatis neither too much nor too little to detrimentally alter the release ofthe active ingredients should be used to form solid oral dosage forms.The amount of disintegrant used varies based upon the type offormulation, and is readily discernible to those of ordinary skill inthe art. Typical pharmaceutical compositions comprise from about 0.5 toabout 15 weight percent of disintegrant, preferably from about 1 toabout 5 weight percent of disintegrant.

Disintegrants that can be used in pharmaceutical compositions and dosageforms include, but are not limited to, agar-agar, alginic acid, calciumcarbonate, microcrystalline cellulose, croscarmellose sodium,crospovidone, polacrilin potassium, sodium starch glycolate, potato ortapioca starch, other starches, pre-gelatinized starch, other starches,clays, other algins, other celluloses, gums, and mixtures thereof.

Lubricants that can be used in pharmaceutical compositions and dosageforms include, but are not limited to, calcium stearate, magnesiumstearate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol,polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate,talc, hydrogenated vegetable oil (e.g., peanut oil, cottonseed oil,sunflower oil, sesame oil, olive oil, corn oil, and soybean oil), zincstearate, ethyl oleate, ethyl laureate, agar, and mixtures thereof.Additional lubricants include, for example, a syloid silica gel(AEROSIL200, manufactured by W.R. Grace Co. of Baltimore, Md.), acoagulated aerosol of synthetic silica (marketed by Degussa Co. ofPlano, Tex.), CAB-O-SIL (a pyrogenic silicon dioxide product sold byCabot Co. of Boston, Mass.), and mixtures thereof. If used at all,lubricants are typically used in an amount of less than about 1 weightpercent of the pharmaceutical compositions or dosage forms into whichthey are incorporated.

In one embodiment, a solid oral dosage form of the invention comprises acompound provided herein, anhydrous lactose, microcrystalline cellulose,polyvinylpyrrolidone, stearic acid, colloidal anhydrous silica, andgelatin.

5.8 Delayed Release Dosage Forms

Active ingredients may be administered by controlled release means or bydelivery devices that are well known to those of ordinary skill in theart. Examples include, but are not limited to, those described in U.S.Pat. Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; and 4,008,719,5,674,533, 5,059,595, 5,591,767, 5,120,548, 5,073,543, 5,639,476,5,354,556, and 5,733,566, each of which is incorporated herein byreference. Such dosage forms can be used to provide slow orcontrolled-release of one or more active ingredients using, for example,hydropropylmethyl cellulose, other polymer matrices, gels, permeablemembranes, osmotic systems, multilayer coatings, microparticles,liposomes, microspheres, or a combination thereof to provide the desiredrelease profile in varying proportions. Suitable controlled-releaseformulations known to those of ordinary skill in the art, includingthose described herein, can be readily selected for use with the activeingredients provided herein. Thus, provided herein are single unitdosage forms suitable for oral administration such as, but not limitedto, tablets, capsules, gelcaps, and caplets that are adapted forcontrolled-release.

All controlled-release pharmaceutical products have a common goal ofimproving drug therapy over that achieved by their non-controlledcounterparts. Ideally, the use of an optimally designedcontrolled-release preparation in medical treatment is characterized bya minimum of drug substance being employed to cure or control thecondition in a minimum amount of time. Advantages of controlled-releaseformulations include extended activity of the drug, reduced dosagefrequency, and increased patient compliance. In addition,controlled-release formulations can be used to affect the time of onsetof action or other characteristics, such as blood levels of the drug,and can thus affect the occurrence of side (e.g., adverse) effects.

Most controlled-release formulations are designed to initially releasean amount of drug (active ingredient) that promptly produces the desiredtherapeutic effect, and gradually and continually release of otheramounts of drug to maintain this level of therapeutic or prophylacticeffect over an extended period of time. In order to maintain thisconstant level of drug in the body, the drug must be released from thedosage form at a rate that will replace the amount of drug beingmetabolized and excreted from the body. Controlled-release of an activeingredient can be stimulated by various conditions including, but notlimited to, pH, temperature, enzymes, water, or other physiologicalconditions or compounds.

5.9 Parenteral Dosage Forms

Parenteral dosage forms can be administered to patients by variousroutes including, but not limited to, subcutaneous, intravenous(including bolus injection), intramuscular, and intraarterial. Becausetheir administration typically bypasses patients' natural defensesagainst contaminants, parenteral dosage forms are preferably sterile orcapable of being sterilized prior to administration to a patient.Examples of parenteral dosage forms include, but are not limited to,solutions ready for injection, dry products ready to be dissolved orsuspended in a pharmaceutically acceptable vehicle for injection,suspensions ready for injection, and emulsions.

Suitable vehicles that can be used to provide parenteral dosage formsare well known to those skilled in the art. Examples include, but arenot limited to: Water for Injection USP; aqueous vehicles such as, butnot limited to, Sodium Chloride Injection, Ringer's Injection, DextroseInjection, Dextrose and Sodium Chloride Injection, and Lactated Ringer'sInjection; water-miscible vehicles such as, but not limited to, ethylalcohol, polyethylene glycol, and polypropylene glycol; and non-aqueousvehicles such as, but not limited to, corn oil, cottonseed oil, peanutoil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

Compounds that increase the solubility of one or more of the activeingredients disclosed herein can also be incorporated into theparenteral dosage forms provided herein. For example, cyclodextrin andits derivatives can be used to increase the solubility of a compound andits derivatives. See, e.g., U.S. Pat. No. 5,134,127, which isincorporated herein by reference.

5.10 Topical and Mucosal Dosage Forms

Topical and mucosal dosage forms provided herein include, but are notlimited to, sprays, aerosols, solutions, emulsions, suspensions, eyedrops or other ophthalmic preparations, or other forms known to one ofskill in the art. See, e.g., Remington's Pharmaceutical Sciences,16^(th) and 18^(th) eds., Mack Publishing, Easton Pa. (1980 & 1990); andIntroduction to Pharmaceutical Dosage Forms, 4th ed., Lea & Febiger,Philadelphia (1985). Dosage forms suitable for treating mucosal tissueswithin the oral cavity can be formulated as mouthwashes or as oral gels.

Suitable excipients (e.g., carriers and diluents) and other materialsthat can be used to provide topical and mucosal dosage forms are wellknown to those skilled in the pharmaceutical arts, and depend on theparticular tissue to which a given pharmaceutical composition or dosageform will be applied. With that fact in mind, typical excipientsinclude, but are not limited to, water, acetone, ethanol, ethyleneglycol, propylene glycol, butane-1,3-diol, isopropyl myristate,isopropyl palmitate, mineral oil, and mixtures thereof to formsolutions, emulsions or gels, which are non-toxic and pharmaceuticallyacceptable. Moisturizers or humectants can also be added topharmaceutical compositions and dosage forms if desired. Examples ofsuch additional ingredients are well known in the art. See, e.g.,Remington's Pharmaceutical Sciences, 16^(th) and 18^(th) eds., MackPublishing, Easton Pa. (1980 & 1990).

The pH of a pharmaceutical composition or dosage form may also beadjusted to improve delivery of one or more active ingredients.Similarly, the polarity of a solvent carrier, its ionic strength, ortonicity can be adjusted to improve delivery. Compounds such asstearates can also be added to pharmaceutical compositions or dosageforms to advantageously alter the hydrophilicity or lipophilicity of oneor more active ingredients so as to improve delivery. In this regard,stearates can serve as a lipid vehicle for the formulation, as anemulsifying agent or surfactant, and as a delivery-enhancing orpenetration-enhancing agent. Different salts, hydrates or solvates ofthe active ingredients can be used to further adjust the properties ofthe resulting composition.

5.11 Kits

In some embodiments provided herein, active ingredients are preferablynot administered to a patient at the same time or by the same route ofadministration. Thus, provided herein are kits which, when used by themedical practitioner, can simplify the administration of appropriateamounts of active ingredients to a patient.

In one embodiment a kit provided herein comprises a compound providedherein, or a pharmaceutically acceptable salt, solvate or hydratethereof. Kits may further comprise additional active agents, includingbut not limited to those disclosed herein.

Kits provided herein may further comprise devices that are used toadminister the active ingredients. Examples of such devices include, butare not limited to, syringes, drip bags, patches, and inhalers.

Kits may further comprise cells or blood for transplantation as well aspharmaceutically acceptable vehicles that can be used to administer oneor more active ingredients. For example, if an active ingredient isprovided in a solid form that must be reconstituted for parenteraladministration, the kit can comprise a sealed container of a suitablevehicle in which the active ingredient can be dissolved to form aparticulate-free sterile solution that is suitable for parenteraladministration. Examples of pharmaceutically acceptable vehiclesinclude, but are not limited to: Water for Injection USP; aqueousvehicles such as, but not limited to, Sodium Chloride Injection,Ringer's Injection, Dextrose Injection, Dextrose and Sodium ChlorideInjection, and Lactated Ringer's Injection; water-miscible vehicles suchas, but not limited to, ethyl alcohol, polyethylene glycol, andpolypropylene glycol; and non-aqueous vehicles such as, but not limitedto, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate,isopropyl myristate, and benzyl benzoate.

5.12 Antibodies

Antibodies that immunospecifically bind to CRBN (anti-CRBN antibodies)provided herein include, but are not limited to, synthetic antibodies,monoclonal antibodies, recombinantly produced antibodies, multispecificantibodies (including bi-specific antibodies), human antibodies,humanized antibodies, chimeric antibodies, intrabodies, single-chain Fvs(scFv) (e.g., including monospecific, bispecific, etc.), camelizedantibodies, Fab fragments, F(ab′) fragments, disulfide-linked Fvs(sdFv), anti-idiotypic (anti-Id) antibodies, and antigen- orepitope-binding fragments of any of the above.

In particular, antibodies provided herein include immunoglobulinmolecules and immunologically active portions of immunoglobulinmolecules, i.e., molecules that contain an antigen binding site thatimmunospecifically binds to a CRBN antigen. The immunoglobulin moleculesprovided herein can be of any type (e.g., IgG, IgE, IgM, IgD, IgA andIgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass ofimmunoglobulin molecule. In a specific embodiment, an antibody providedherein is an IgG antibody, and, in certain embodiments, an IgG1 or IgG4.

Also provided herein is an isolated CRBN antibody, for example,“CRBN70,” as prepared according to Example 6.20 or 6.21 below. In oneembodiment, the antibody is a polyclonal antibody. In anotherembodiment, the antibody is a monoclonal antibody. In some embodiments,the antibody is a rabbit polyclonal antibody. In other embodiments, theantibody is a rabbit monoclonal antibody.

In another embodiment, provided herein is an isolated antibody whichimmunospecifically binds to the epitope having an amino acid sequenceEEFHGRTLHDDDC (SEQ ID:1). In another embodiment, the antibodyimmunospecifically binds to the epitope having an amino acid sequenceEEFHGRTLHDDDC (SEQ ID:1), wherein the peptide is coupled to KeyholeLimpet Hemocyanin (KLH). In one embodiment, the antibody is a polyclonalantibody. In another embodiment, the antibody is a monoclonal antibody.In some embodiments, the antibody is a rabbit polyclonal antibody. Inother embodiments, the antibody is a rabbit monoclonal antibody. Incertain embodiments, the antibody immunospecifically binds peptide 65-76(SEQ ID NO:1) of human CRBN (SEQ ID NO:12).

In certain embodiments, provided herein is an antibody thatimmunospecifically binds CRBN and comprises a heavy chain having theamino acid sequence depicted in SEQ ID NO:5, or the VH domain, VH CDR1,VH CDR2, and/or VH CDR3 thereof. In other embodiment, the antibodyimmunospecifically binds CRBN and comprises a light chain having theamino acid sequence depicted in SEQ ID NO:7, or the VL domain, VL CDR1,VL CDR2, and/or VL CDR3 thereof. In some embodiments, the antibodycomprises a heavy chain having the amino acid sequence depicted in SEQID NO:5, or the VH domain, VH CDR1, VH CDR2, and/or VH CDR3 thereof; anda light chain having the amino acid sequence depicted in SEQ ID NO:7, orthe VL domain, VL CDR1, VL CDR2, and/or VL CDR3 thereof. In certainembodiments, the antibody immunospecifically binds CRBN and comprises aheavy chain having the amino acid sequence depicted in SEQ ID NO:9, orthe VH domain, VH CDR1, VH CDR2, and/or VH CDR3 thereof. In otherembodiment, the antibody immunospecifically binds CRBN and comprises alight chain having the amino acid sequence depicted in SEQ ID NO:11, orthe VL domain, VL CDR1, VL CDR2, and/or VL CDR3 thereof. In someembodiments, the antibody comprises a heavy chain having the amino acidsequence depicted in SEQ ID NO:9, or the VH domain, VH CDR1, VH CDR2,and/or VH CDR3 thereof; and a light chain having the amino acid sequencedepicted in SEQ ID NO:11, or the VL domain, VL CDR1, VL CDR2, and/or VLCDR3 thereof. In certain embodiments, the antibody immunospecificallybinds peptide 65-76 (SEQ ID NO:1) of human CRBN (SEQ ID NO:12).

Any of the CRBN antibodies provided herein can be used in any of themethods provided herein

Variants and derivatives of antibodies include antibody fragments thatretain the ability to specifically bind to an epitope. Exemplaryfragments include Fab fragments (an antibody fragment that contains theantigen-binding domain and comprises a light chain and part of a heavychain bridged by a disulfide bond); Fab′ (an antibody fragmentcontaining a single anti-binding domain comprising an Fab and anadditional portion of the heavy chain through the hinge region); F(ab′)₂(two Fab′ molecules joined by interchain disulfide bonds in the hingeregions of the heavy chains; the Fab′ molecules may be directed towardthe same or different epitopes); a bispecific Fab (a Fab molecule havingtwo antigen binding domains, each of which may be directed to adifferent epitope); a single chain Fab chain comprising a variableregion, also known as, a sFv (the variable, antigen-bindingdeterminative region of a single light and heavy chain of an antibodylinked together by a chain of 10-25 amino acids); a disulfide-linked Fv,or dsFv (the variable, antigen-binding determinative region of a singlelight and heavy chain of an antibody linked together by a disulfidebond); a camelized VH (the variable, antigen-binding determinativeregion of a single heavy chain of an antibody in which some amino acidsat the VH interface are those found in the heavy chain of naturallyoccurring camel antibodies); a bispecific sFv (a sFv or a dsFv moleculehaving two antigen-binding domains, each of which may be directed to adifferent epitope); a diabody (a dimerized sFv formed when the VH domainof a first sFv assembles with the VL domain of a second sFv and the VLdomain of the first sFv assembles with the VH domain of the second sFv;the two antigen-binding regions of the diabody may be directed towardsthe same or different epitopes); and a triabody (a trimerized sFv,formed in a manner similar to a diabody, but in which threeantigen-binding domains are created in a single complex; the threeantigen binding domains may be directed towards the same or differentepitopes). Derivatives of antibodies also include one or more CDRsequences of an antibody combining site. The CDR sequences may be linkedtogether on a scaffold when two or more CDR sequences are present. Incertain embodiments, the anti-CRBN antibody comprises a single-chain Fv(“scFv”). scFvs are antibody fragments comprising the VH and VL domainsof an antibody, wherein these domains are present in a singlepolypeptide chain. Generally, the scFv polypeptide further comprises apolypeptide linker between the VH and VL domains which enables the scFvto form the desired structure for antigen binding. For a review of scFvssee Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994).

Provided herein are antibodies that immunospecifically bind to a CRBNepitope, the antibodies comprising derivatives of the VH and VL chainsdescribed herein that immunospecifically bind to a CRBN antigen or aCRBN epitope. Standard techniques known to those of skill in the art canbe used to introduce mutations in the nucleotide sequence encoding amolecule of the invention, including, for example, site-directedmutagenesis and PCR-mediated mutagenesis which results in amino acidsubstitutions. Preferably, the derivatives include less than 25 aminoacid substitutions, less than 20 amino acid substitutions, less than 15amino acid substitutions, less than 10 amino acid substitutions, lessthan 5 amino acid substitutions, less than 4 amino acid substitutions,less than 3 amino acid substitutions, or less than 2 amino acidsubstitutions relative to the original molecule. In a preferredembodiment, the derivatives have conservative amino acid substitutionsare made at one or more predicted non-essential amino acid residues. A“conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a side chain witha similar charge. Families of amino acid residues having side chainswith similar charges have been defined in the art. These familiesinclude amino acids with basic side chains (e.g., lysine, arginine,histidine), acidic side chains (e.g., aspartic acid, glutamic acid),uncharged polar side chains (e.g., glycine, asparagine, glutamine,serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g.,alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Alternatively, mutations can beintroduced randomly along all or part of the coding sequence, such as bysaturation mutagenesis, and the resultant mutants can be screened forbiological activity to identify mutants that retain activity. Followingmutagenesis, the encoded protein can be expressed and the activity ofthe protein can be determined.

In another embodiment, an antibody that immunospecifically binds to aCRBN epitope comprises an amino acid sequence that is at least 35%, atleast 40%, at least 45%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, or at least 99% identical to the amino acidsequence of CGN-6-1-11 OR CGN-6-1-11, or an antigen-binding fragmentthereof, such as a VH domain, VL domain, VH chain, or VL chain. In oneembodiment, an antibody that immunospecifically binds to a CRBN epitopecomprises an amino acid sequence that is at least 35%, at least 40%, atleast 45%, at least 50%, at least 55%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, or at least 99% identical to an amino acid sequence depictedin SEQ ID NOS:5, 7, 9 or 11.

In a specific embodiment, an antibody that immunospecifically binds aCRBN antigen comprises an amino acid sequence of a VH chain and/or anamino acid sequence a VL chain encoded by a nucleotide sequence thathybridizes to (1) the complement of a nucleotide sequence encoding anyone of the VH and/or VL chains depicted in SEQ ID NOS:5 or 9 (H chain)and/or SEQ ID NOS:7 or 11 (L chain) under stringent conditions (e.g.,hybridization to filter-bound DNA in 6× sodium chloride/sodium citrate(SSC) at about 45° C. followed by one or more washes in 0.2×SSC/0.1% SDSat about 50-65° C.) under highly stringent conditions (e.g.,hybridization to filter-bound nucleic acid in 6×SSC at about 45° C.followed by one or more washes in 0.1×SSC/0.2% SDS at about 68° C.), orunder other stringent hybridization conditions which are known to thoseof skill in the art (see, for example, Ausubel, F. M. et al., eds.,1989, Current Protocols in Molecular Biology, Vol. I, Green PublishingAssociates, Inc. and John Wiley & Sons, Inc., New York at pages6.3.1-6.3.6 and 2.10.3).

The anti-CRBN antibodies may be from any animal origin including birdsand mammals (e.g., human, murine, donkey, sheep, rabbit, goat, guineapig, camel, horse, or chicken). In certain embodiments, the anti-CRBNantibodies are human or humanized monoclonal antibodies. As used herein,“human” antibodies include antibodies having the amino acid sequence ofa human immunoglobulin and include antibodies isolated from humanimmunoglobulin libraries or from mice that express antibodies from humangenes. In certain embodiments, the anti-CRBN antibodies are fully humanantibodies, such as fully human antibodies that immunospecifically binda CRBN polypeptide, a CRBN polypeptide fragment, or a CRBN epitope. Suchfully human antibodies would be advantageous over fully mouse (or otherfull or partial non-human species antibodies), humanized antibodies, orchimeric antibodies to minimize the development of unwanted or unneededside effects, such as immune responses directed toward non-fully humanantibodies (e.g., anti-CRBN antibodies derived from other species) whenadministered to the subject. Anti-CRBN antibodies provided herein may bemonospecific, bispecific, trispecific or of greater multispecificity.Multispecific antibodies may be specific for different epitopes of aCRBN polypeptide or may be specific for both a CRBN polypeptide as wellas for a heterologous epitope, such as a heterologous polypeptide orsolid support material. In certain embodiments, the antibodies providedherein are monospecific for a given epitope of a CRBN polypeptide and donot immunospecifically bind to other epitopes. In certain embodiments,provided herein are anti-CRBN antibodies that immunospecifically bind toa CRBN epitope (e.g., EEFHGRTLHDDD (residues 1-12 of SEQ ID NO:1) orpeptide 65-76 of human CRBN (SEQ ID NO:12)) or a CRBN antigen, as wellas methods of use thereof.

Standard techniques known to those of skill in the art can be used tointroduce mutations in the nucleotide sequence encoding an anti-CRBNprovided herein, including, for example, site-directed mutagenesis andPCR-mediated mutagenesis which results in amino acid substitutions. A“conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a side chain witha similar charge. Families of amino acid residues having side chainswith similar charges have been defined in the art. These familiesinclude amino acids with basic side chains (e.g., lysine, arginine,histidine), acidic side chains (e.g., aspartic acid, glutamic acid),uncharged polar side chains (e.g., glycine, asparagine, glutamine,serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g.,alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Alternatively, mutations can beintroduced randomly along all or part of the coding sequence, such as bysaturation mutagenesis, and the resultant mutants can be screened forbiological activity to identify mutants that retain activity. Followingmutagenesis, the encoded protein can be expressed and the activity ofthe protein can be determined.

In some embodiments, the antibody is a fully human anti-human CRBNantibody, such as a fully human monoclonal antibody. Fully humanantibodies may be produced by any method known in the art. Exemplarymethods include immunization with a CRBN antigen (any CRBN polypeptidecapable of eliciting an immune response, and optionally conjugated to acarrier) of transgenic animals (e.g., mice) that are capable ofproducing a repertoire of human antibodies in the absence of endogenousimmunoglobulin production; see, e.g., Jakobovits et al., (1993) Proc.Natl. Acad. Sci., 90:2551; Jakobovits et al., (1993) Nature, 362:255 258(1993); Bruggermann et al., (1993) Year in Immunol., 7:33. Other methodsof producing anti-CRBN antibodies can be found in the Examples providedherein.

Alternatively, fully human antibodies may be generated through the invitro screening of phage display antibody libraries; see e.g.,Hoogenboom et al., J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol.Biol., 222:581 (1991), incorporated herein by reference. Variousantibody-containing phage display libraries have been described and maybe readily prepared by one skilled in the art. Libraries may contain adiversity of human antibody sequences, such as human Fab, Fv, and scFvfragments, that may be screened against an appropriate target.

The anti-CRBN antibodies include antibodies that are chemicallymodified, i.e., by the covalent attachment of any type of molecule tothe antibody. For example, but not by way of limitation, the antibodyderivatives include antibodies that have been chemically modified, e.g.,by glycosylation, acetylation, pegylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, linkage to a cellular ligand or other protein, etc. Any ofnumerous chemical modifications may be carried out by known techniques,including, but not limited to specific chemical cleavage, acetylation,formulation, metabolic synthesis of tunicamycin, etc. Additionally, theantibody may contain one or more non-classical amino acids.

In certain embodiments, anti-CRBN antibodies that immunospecificallybind to a CRBN antigen comprise a framework region known to those ofskill in the art (e.g., a human or non-human fragment). The frameworkregion may, for example, be naturally occurring or consensus frameworkregions. In some embodiments, the framework region of an anti-CRBNantibody is human (see, e.g., Chothia et al., 1998, J. Mol. Biol.278:457-479 for a listing of human framework regions, which isincorporated by reference herein in its entirety). See also Kabat et al.(1991) Sequences of Proteins of Immunological Interest (U.S. Departmentof Health and Human Services, Washington, D.C.) 5th ed.

In certain embodiments, the anti-CRBN antibodies provided herein arechimeric or humanized antibodies. In some embodiments, the antibodiesprovided herein comprise human framework regions with one or more aminoacid substitutions at one, two, three or more of the following residues:(a) rare framework residues that differ between the murine antibodyframework (i.e., donor antibody framework) and the human antibodyframework (i.e., acceptor antibody framework); (b) Venier zone residueswhen differing between donor antibody framework and acceptor antibodyframework; (c) interchain packing residues at the VH/VL interface thatdiffer between the donor antibody framework and the acceptor antibodyframework; (d) canonical residues which differ between the donorantibody framework and the acceptor antibody framework sequences,particularly the framework regions crucial for the definition of thecanonical class of the murine antibody CDR loops; (e) residues that areadjacent to a CDR; (g) residues capable of interacting with the antigen;(h) residues capable of interacting with the CDR; and (i) contactresidues between the VH domain and the VL domain. In certainembodiments, antibodies that immunospecifically bind to a CRBN antigencomprising the human framework regions with one or more amino acidsubstitutions at one, two, three or more of the above-identifiedresidues are antagonistic CRBN antibodies.

In other embodiments, fusion proteins comprising an anti-CRBN antibodyare provided herein that immunospecifically binds to a CRBN antigen anda heterologous polypeptide.

5.13 Diagnostic Use of Antibodies

Labeled CRBN antibodies provided herein and derivatives and analogsthereof, which immunospecifically bind to a CRBN antigen can be used fordiagnostic purposes to detect, diagnose, or monitor CRBN expressionlevels or a CRBN-mediated disease in a patient.

In some embodiments, provided herein are methods of utilizing a CRBNantibody to measure expression levels of CRBN in patient tumor or hostcells, to predict clinical response to therapy with thalidomide,lenalidomide, pomalidomide, or3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione, astereoisomer thereof, or a pharmaceutically acceptable salt, solvate,hydrate, co-crystal, clathrate, or polymorph thereof. Also providedherein are methods of utilizing a CRBN antibody provided herein tomeasure expression levels of CRBN in patient tumor or host cells, tomonitor clinical response to therapy with thalidomide, lenalidomide,pomalidomide, or3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione, astereoisomer thereof, or a pharmaceutically acceptable salt, solvate,hydrate, co-crystal, clathrate, or polymorph thereof. Also providedherein are methods of utilizing a CRBN antibody provided herein tomeasure expression levels of CRBN in patient tumor or host cells, tomonitor patient compliance to dosing to therapy with thalidomide,lenalidomide, pomalidomide, or3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione, astereoisomer thereof, or a pharmaceutically acceptable salt, solvate,hydrate, co-crystal, clathrate, or polymorph thereof. Also providedherein are methods of utilizing a CRBN antibody provided herein tomeasure expression levels of CRBN in patient tumor or host cells, tomonitor development of resistance to therapy with thalidomide,lenalidomide, pomalidomide, or3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione, astereoisomer thereof, or a pharmaceutically acceptable salt, solvate,hydrate, co-crystal, clathrate, or polymorph thereof.

Provided herein are diagnostic assays for diagnosing a CRBN-mediateddisease comprising: (a) assaying for the level of a CRBN antigen incells or a tissue sample of an individual using one or more antibodiesof the invention that immunospecifically bind to a CRBN antigen; and (b)comparing the level of the CRBN antigen with a control level, e.g.,levels in normal tissue samples, whereby an increase in the assayed CRBNantigen level compared to the control level of the CRBN antigen isindicative of a CRBN-mediated disease.

Antibodies provided herein can be used to assay CRBN antigen levels in abiological sample using classical immunohistological methods asdescribed herein or as known to those of skill in the art (e.g., seeJalkanen et al., 1985, J. Cell. Biol. 101:976-985; and Jalkanen et al.,1987, J. Cell. Biol. 105:3087-3096). Other antibody-based methods usefulfor detecting protein gene expression include immunoassays, such as theenzyme linked immunosorbent assay (ELISA) and the radioimmunoassay(RIA). Suitable antibody assay labels are known in the art and includeenzyme labels, such as, glucose oxidase; radioisotopes, such as iodine(¹²⁵I, ¹²¹I), carbon (¹⁴C) sulfur (³⁵S), tritium (³H), indium (¹²¹In),and technetium (⁹⁹Tc); luminescent labels, such as luminol; andfluorescent labels, such as fluorescein and rhodamine, and biotin.

Provided herein are methods for the detection or CRBN in a patient. Inone embodiment, the method comprises: a) administering (for example,parenterally, subcutaneously, or intraperitoneally) to a subject aneffective amount of a labeled antibody that immunospecifically binds toa CRBN antigen; b) waiting for a time interval following theadministering for permitting the labeled antibody to preferentiallyconcentrate at sites in the subject where the CRBN antigen is expressed(and for unbound labeled molecule to be cleared to background level); c)determining background level; and d) detecting the labeled antibody inthe subject, such that detection of labeled antibody above thebackground level indicates that the subject has increased CRBNexpression. Background level can be determined by various methodsincluding, comparing the amount of labeled molecule detected to astandard value previously determined for a particular system.

It will be understood in the art that the size of the subject and theimaging system used will determine the quantity of imaging moiety neededto produce diagnostic images. In the case of a radioisotope moiety, fora human subject, the quantity of radioactivity injected will normallyrange from about 5 to 20 millicuries of ⁹⁹Tc. The labeled antibody willthen preferentially accumulate at the location of cells which containthe specific protein. In vivo tumor imaging is described in S. W.Burchiel et al., “Immunopharmacokinetics of Radiolabeled Antibodies andTheir Fragments.” (Chapter 13 in Tumor Imaging: The RadiochemicalDetection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., MassonPublishing Inc. (1982).

Depending on several variables, including the type of label used and themode of administration, the time interval following the administrationfor permitting the labeled antibody to preferentially concentrate atsites in the subject and for unbound labeled antibody to be cleared tobackground level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. Inanother embodiment the time interval following administration is 5 to 20days or 5 to 10 days.

Presence of the labeled molecule can be detected in the subject usingmethods known in the art for in vivo scanning. These methods depend uponthe type of label used. Skilled artisans will be able to determine theappropriate method for detecting a particular label. Methods and devicesthat may be used in the diagnostic methods of the invention include, butare not limited to, computed tomography (CT), whole body scan such asposition emission tomography (PET), magnetic resonance imaging (MRI),and sonography.

In a specific embodiment, the molecule is labeled with a radioisotopeand is detected in the patient using a radiation responsive surgicalinstrument (Thurston et al., U.S. Pat. No. 5,441,050). In anotherembodiment, the molecule is labeled with a fluorescent compound and isdetected in the patient using a fluorescence responsive scanninginstrument. In another embodiment, the molecule is labeled with apositron emitting metal and is detected in the patient using positronemission-tomography. In yet another embodiment, the molecule is labeledwith a paramagnetic label and is detected in a patient using magneticresonance imaging (MRI).

6. EXAMPLES

Certain embodiments of the invention are illustrated by the followingnon-limiting examples.

6.1 Preparation of3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione(lenalidomide) Methyl 2-bromomethyl-3-nitrobenzoate

A stirred mixture of methyl 2-methyl-3-nitrobenzoate (14.0 g, 71.7 mmol)and N-bromosuccinimide (15.3 g, 86.1 mmol) in carbon tetrachloride (200mL) was heated under gentle reflux for 15 hours while a 100 W bulbsituated 2 cm away was shining on the flask. The mixture was filteredand the solid was washed with methylene chloride (50 mL). The filtratewas washed with water (2×100 mL), brine (100 mL) and dried. The solventwas removed in vacuo and the residue was purified by flashchromatography (hexane/ethyl acetate, 8/2) to afford 19 g (96%) of theproduct as a yellow solid: mp 70.0-71.5° C.; 1H NMR (CDCl₃) δ 8.12-8.09(dd, J=1.3 and 7.8 Hz, 1H), 7.97-7.94 (dd, J=1.3 and 8.2 Hz, 1H), 7.54(t, J=8.0 Hz, 1H). 5.15 (s, 2H), 4.00 (s, 3H); ¹³C NMR (CDCl₃) δ 165.85,150.58, 134.68, 132.38, 129.08, 127.80, 53.06, 22.69; HPLC, WaterNove-Pak/C18, 3.9×150 mm, 4 micron, 1 mL/min, 240 nm, 40/60 CH₃CN/0.1%H₃PO₄ (aq) 7.27 min(98.92%); Anal. Calcd for C₉H₈NO₄Br: C, 39.44; H,2.94; N, 5.11; Br, 29.15. Found: C, 39.46; H, 3.00; N, 5.00; Br, 29.11.

t-Butyl N-(1-oxo-4-nitroisoindolin-2-yl)-L-glutamine

Triethylamine (2.9 g, 28.6 mmol) was added dropwise to a stirred mixtureof methyl 2-bromomethyl-3-nitrobenzoate (3.5 g, 13.0 mmol) andL-glutamine t-butyl ester hydrochloride (3.1 g, 13.0 mmol) intetrahydrofuran (90 mL). The mixture was heated to reflux for 24 hours.To the cooled mixture was added methylene chloride (150 mL) and themixture was washed with water (2×40 mL), brine (40 mL) and dried. Thesolvent was removed in vacuo and the residue was purified by flashchromatography (3% CH₃OH in methylene chloride) to afford 2.84 g (60%)of crude product which was used directly in the next reaction: 1H NMR(CDCl₃) δ 8.40 (d, J=8.1 Hz, 1H), 8.15 (d, J=7.5 Hz, 1H), 7.71 (t, J=7.8Hz, 1H), 5.83 (s, 1H), 5.61 (s, 1H), 5.12 (d, J=19.4 Hz, 1H), 5.04-4.98(m, 1H), 4.92 (d, J=19.4 Hz, 1H), 2.49-2.22 (m, 4H). 1.46 (s, 9H); HPLC,Waters Nova-Pak C18, 3.9×150 mm, 4 micron, 1 mL/min, 240 nm, 25/75CH₃CN/0.1% H₃PO₄ (aq) 6.75 min(99.94%).

N-(1-oxo-4-nitroisoindolin-2-yl)-L-glutamine

Hydrogen chloride gas was bubbled into a stirred 5° C. solution oft-butyl N-(1-oxo-4-nitro-isoindolin-2-yl)-L-glutamine (3.6 g, 9.9 mmol)in methylene chloride (60 mL) for 1 hour. The mixture was then stirredat room temperature for another hour. Ether (40 mL) was added and theresulting mixture was stirred for 30 minutes. The slurry was filtered,washed with ether and dried to afford 3.3 g of the product: 1H NMR(DMSO-d₆) δ 8.45 (d, J=8.1 Hz, 1H), 8.15 (d, J=7.5 Hz, 1H), 7.83 (t,J=7.9 Hz. 1H), 7.24 (s, 1H), 6.76 (s, 1H), 4.93 (s, 2H), 4.84-4.78 (dd,J=4.8amd 10.4 Hz, 1H), 2.34-2.10 (m, 4H); ¹³C NMR (DMSO-d₆) δ 173.03,171.88, 165.96, 143.35, 137.49, 134.77, 130.10, 129.61, 126.95, 53.65,48.13, 31.50, 24.69; Anal. Calcd for C₁₃H₁₃N₃O₆: C, 50.82; H, 4.26; N,13.68. Found: C, 50.53; H. 4.37; N, 13.22.

(S)-3-(1-oxo-4-nitroisoindolin-2-yl)piperidine-2,6-dione

A stirred suspension mixture ofN-(1-oxo-4-nitroisoindolin-2-yl)-L-glutamine (3.2 g, 10.5 mmol) inanhydrous methylene chloride (150 mL) was cooled to −40° C. withisopropanol/dry ice bath. Thionyl chloride (0.82 mL, 11.3 mmol) wasadded dropwise to the cooled mixture followed by pyridine (0.9 g. 11.3mmol). After 30 min, triethylamine (1.2 g, 11.5 mmol) was added and themixture was stirred at −30 to −40° C. for 3 hours. The mixture waspoured into ice water (200 mL) and the aqueous layer was extracted withmethylene chloride (40 mL). The methylene chloride solution was washedwith water (2×60 mL), brine (60 mL) and dried. The solvent was removedin vacuo and the solid residue was slurried with ethyl acetate (20 mL)to give 2.2 g (75%) of the product as a white solid: mp 285° C.; 1H NMR(DMSO-d₆) δ : 1.04 (s, 1H), 8.49-8.45 (dd, J=0.8 and 8.2 Hz, 1H),8.21-8.17 (dd, J=7.3 Hz, 1H), 7.84 (t, J=7.6 Hz, 1H), 5.23-5.15 (dd,J=4.9 and 13.0 Hz, 1H), 4.96 (dd, J=19.3 and 32.4 Hz, 2H), 3.00-2.85 (m,1H), 2.64-2.49 (m, 2H), 2.08-1.98 (m, 1H); ¹³C NMR (DMSO-d₆) δ 172.79,170.69, 165.93, 143.33, 137.40, 134.68, 130.15, 129.60, 127.02, 51.82,48.43, 31.16. 22.23; HPLC, Waters Nove-Pak/C18, 3.9×150 mm, 4 micron, 1mL/min, 240 nm, 20/80 CH₃CN/0.1% H₃PO₄ (aq) 3.67 min(100%); Anal. Calcdfor C₁₃H_(n)N₃O₅: C, 53.98; H, 3.83; N, 14.53. Found: C, 53.92; H, 3.70;N, 14.10.

3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione

A mixture of (S)-3-(1-oxo-4-nitroisoindolin-2-yl)piperidine-2,6-dione(1.0 g, 3.5 mmol) and 10% Pd/C (0.3 g) in methanol (600 mL) washydrogenated in a Parr-Shaker apparatus at 50 psi of hydrogen for 5hours. The mixture was filtered through Celite and the filtrate wasconcentrated in vacuo. The solid was slurried in hot ethyl acetate for30 min, filtered and dried to afford 0.46 g (51%) of the product as awhite solid: mp 235.5-239° C.; ¹H NMR (DMSO-d₆) δ 11.01 (s, 1H). 7.19(t, J=7.6 Hz, 1H). 6.90 (d. J=7.3 Hz, 1H), 6.78 (d, J=7.8 Hz, 1H), 5.42(s, 2H). 5.12 (dd. J=5.1 and 13.1 Hz, 1H), 4.17 (dd, J=17.0 and 28.8 Hz,2H), 2.92-2.85 (m, 1H). 2.64-2.49 (m, 1H). 2.34-2.27 (m, 1H), 2.06-1.99(m, 1H); ¹³C NMR (DMSO-d₆) δ 172.85, 171.19, 168.84, 143.58, 132.22.128.79, 125.56, 116.37, 110.39, 51.48, 45.49, 31.20, 22.74; HPLC. WatersNova-Pak/C18, 3.9×150 mm, 4 micron, 1 mL/min, 240 nm, 10/90 CH₃CN/0.1%H₃PO₄ (aq) 0.96 min(100%); Chiral analysis, Daicel Chiral Pak AD, 40/60Hexane/IPA, 6.60 min(99.42%); Anal. Calcd for C₁₃H₁₃N₃O₃: C, 60.23; H,5.05; N, 16.21. Found: C, 59.96; H. 4.98; N, 15.84.

3-(4-Amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione mayalso be prepared by methods known in the art, for example, as providedin Drugs of the Future, 2003, 28(5): 425-431, the entirety of which isincorporated by reference.

6.2 Preparation of4-amino-2-(2,6-dioxopiperidin-3-yl)-1H-isoindole-1,3-dione(pomalidomide)

The preparation of4-amino-2-(2,6-dioxopiperidin-3-yl)-1H-isoindole-1,3-dione is described,for example, in U.S. Pat. Nos. 7,812,169 and 7,709,502, the entirety ofeach of which is incorporated by reference.

Into a stirring solution of carboxybenzyloxy-L-glutamine (2.8 g, 10mmols) in 40 mL anhydrous THF, 1,1-carbonyldiimidazole (1.92 g, 12mmols) were added. The reaction mixture was heated under reflux for 18hours. The THF was evaporated and the product was dissolved inchloroform. The chloroform layer was washed with water and brine anddried over anhydrous CaSO₄, filtered and evaporated to give white solid.The solid product was crystallized from ethyl ether to give 2.4 gramscrystalline powder (90%). (Alternatively, carboxybenzyloxy-L-glutaminecan be cyclized by treating with SOCl₂ in N,N-dimethylformamide at −70°C. to 0° C. for 1 hour to form the product). The reaction mixture wasdiluted with CHCl₃ and washed with 5% Na₂CO₃, dried over anhydrousNa₂SO₄, filtered, and evaporated to give 2.5 g (90% yield)S(−)-(3-benzyloxycarbonylamino)-glutarimide). ¹H NMR (CDCl₃) δ 8.2 (1H,s broad), 7.4 (5H, s, aromatic), 5.8 (1H, d), 5.15 (2H, s), 4.4 (1H, dd,J=4.5, 3), 2.95-2.4 (3H, m), 1.86 (1H, d, t, J=11.5, 6.5). m.p. 122-124°C. (lit. 122-124° C.).

Into a solution of S(−)-(2-benzyloxycarbonylamino)glutarimide (1.2 g,4.6 mmols) in 15 mL acetic acid glacial, 8 mL of 30% HBr/acetic acidsolution was added at 20° C. The temperature of reaction mixture wasraised to RT and stirred for 1 hour. White solid powder ofS-(−)-2-amino-glutarimide HBr started appearing in reaction mixture. Thesolid was filtered and washed with 5 mL acetic acid glacial and thenwith ether to give 1.8 g (80%) product. Analysis on polarimeter ofproduct showed (−) rotation, [a]²⁵ _(D) (c=1, water)=−37.5° andconfirmed the product as S-(−)-2-amino-glutarimide. ¹H NMR in DMSO-D₆confirmed the product as 2-amino-L-glutarimide HBr.

Into a solution of (4.18 g, 20 mmols S-(−)-2-amino-glutarimide HBr in 50mL of anhydrous DMF, 3.8 g (20 mmols) of 3-nitrophthalic anhydride wasadded. After adding 100 mL acetic acid (glacial), the reaction mixturewas heated at about 70° C. to about 80° C. for about 24 hours.Thereafter, the solvents were evaporated under vacuum to yield anoff-white solid. On adding 10 mL ethyl alcohol to the solid, anoff-white powder product was formed. The product was separated andwashed with 20 mL ethyl alcohol. ¹H NMR (DMSO-D₆) δ 11.25 (1H, s broad),8.35 (1H, d, J=7.2), 8.25 (1H, d, J=7.0), 8.15 (1H, t, J=8.0), 5.2 (1H,dd, J=5.5, 7.2), 3.00-2.85 (1H, m), 2.65-2.4 (2H, m), 2.15-2.05 (1H, m).m.p.: 228-229° C. (lit. 228.5-229.5° C.).

4-Nitro-thalidomide (1 g, 3.3 mmols) was dissolved in 50 mLdioxane/methanol 4:1 mixture and hydrogenated in a Parr hydrogenater at40 psi of hydrogen in the presence of a Pd/C 5% catalyst for about 4hours. After filtering the reaction mixture through a Celite filteringagent, the solvents were evaporated under vacuum to yield a yellowpowder. The product was recrystallized from ethyl acetate/dioxane toyield 800 mg (85% purity) of S(−)-4-amino-thalidomide. ¹H NMR inDMSO-D₆: 11.10 (1H, s broad), 7.45 (1H, t, J=7.5), 7.05 (1H, d, J=5.2),6.95 (1H, d, J=5.2), 6.5 (2H, s broad), 5.05 (1H, dd, J=5.0, 13.42),2.95-2.80 (1H, m), 2.65-2.5 (2H, m), 2.05-1.95 (1H, m). m.p.318.2-319.5° C. Absolute configuration was determined by comparison ofspecific rotation [a]²⁵ _(D) of (R)- and(S)-4-amino-2-(2,6-dioxopiperidin-3-yl)-1H-isoindole-1,3-dione to theanalogous compounds R(+)- and S(−)-thalidomide. Analysis on polarimeterof product showed (−) rotation, [a]²⁵ _(D) (C=0.5, dioxane)=−27.70° andconfirmed the product asS(−)-4-amino-2-(2,6-dioxopiperidin-3-yl)-1H-isoindole-1,3-dione.

The two enantiomers were resolved by chiral HPLC column Welk-01 (10mm×750 mm) and eluted with CH3CN/MeOH/H20 1:1:5 mixture. The retentiontime for the S(−) enantiomer was 33.74 minutes and for the R(+)enantiomer 35.62 minutes at a flow rate of 2 mL/min at 240 nm,respectively.

6.3 Preparation of3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione(Compound B)

To a solution of potassium hydroxide (16.1 g, 286 mmol) in water (500mL), was added 3-nitrophthalimide (25.0 g, 130 mmol) in portion at 0° C.The suspension was stirred at 0° C. for 3 hrs, and then heated to 30° C.for 3 hrs. To the solution, was added HCl (100 mL, 6N). The resultingsuspension was cooled to 0° C. for 1 hr. The suspension was filtered andwashed with cold water (2×10 mL) to give 3-nitro-phthalamic acid as awhite solid (24.6 g, 90% yield): ¹H NMR (DMSO-d₆) δ 7.69 (brs, 1H, NHH),7.74 (t, J=8 Hz, 1H, Ar), 7.92 (dd, J=1, 8 Hz, 1H, Ar), 8.13 (dd, J=1, 8Hz, 1H, Ar), 8.15 (brs, 1H, NHH), 13.59 (s, 1H, OH); ¹³C NMR (DMSO-d₆) δ125.33, 129.15, 130.25, 132.54, 136.72, 147.03, 165.90, 167.31.

To a mixture of 3-nitro-phthalamic acid (24.6 g, 117 mmol) and potassiumhydroxide (6.56 g, 117 mmol) in water (118 mL), was added a mixture ofbromine (6 mL), potassium hydroxide (13.2 g, 234 mmol) in water (240 mL)at 0° C., followed by addition of a solution of potassium hydroxide(19.8 g, 351 mmol) in water (350 mL). After 5 minutes at 0° C., themixture was heated in a 100° C. oil bath for 1 hr. The reaction solutionwas cooled to room temperature, and then, in an ice-water bath for 30minutes. To the mixture, a solution of HCl (240 mL, 2N) was addeddropwise at 0° C., and the resulting mixture was kept for 1 hr. Thesuspension was filtered and washed with water (5 mL) to give2-amino-6-nitro-benzoic acid as yellow solid (15.6 g, 73% yield): HPLC:Waters Symmetry C₁₈, 5 μm, 3.9×150 mm, 1 mL/min, 240 nm, CH₃CN/0.1%H₃PO₄, 5% grad to 95% over 5 min, 5.83 min (85%); ¹H NMR (DMSO-d₆) δ6.90 (dd, J=1, 8 Hz, 1H, Ar), 7.01 (dd, J=1, 9 Hz, 1H, Ar), 7.31 (t, J=8Hz, 1H, Ar), 8.5-9.5 (brs, 3H, OH, NH₂); ¹³C NMR (DMSO-d₆) δ 105.58,110.14, 120.07, 131.74, 149.80, 151.36, 166.30; LCMS: MH=183.

A mixture of 2-amino-6-nitro-benzoic acid (1.5 g, 8.2 mmol) in aceticanhydride (15 mL) was heated at 200° C. for 30 minutes in a microwaveoven. The mixture was filtered and washed with ethyl acetate (20 mL).The filtrate was concentrated in vacuo. The solid was stirred in ether(20 mL) for 2 hrs. The suspension was filtered and washed with ether (20mL) to give 2-methyl-5-nitro-benzo[d][1,3]oxazin-4-one as a light brownsolid (1.4 g, 85% yield): HPLC: Waters Symmetry C₁₈, 5 μm, 3.9×150 mm, 1mL/min, 240 nm, CH₃CN/0.1% H₃PO₄, 5% grad 95% in 5 min, 5.36 min (92%);¹H NMR (DMSO-d₆) δ 2.42 (s, 3H, CH₃), 7.79 (dd, J=1, 8 Hz, 1H, Ar), 7.93(dd, J=1, 8 Hz, 1H, Ar), 8.06 (t, J=8 Hz, 1H, Ar); ¹³C NMR (DMSO-d₆) δ20.87, 107.79, 121.54, 128.87, 137.19, 147.12, 148.46, 155.18, 161.78;LCMS: MH=207.

Two vials each with a suspension of5-nitro-2-methyl-benzo[d][1,3]oxazin-4-one (0.60 g, 2.91 mmol) and3-amino-piperidine-2,6-dione hydrogen chloride (0.48 g, 2.91 mmol) inpyridine (15 mL) were heated at 170° C. for 10 minutes in a microwaveoven. The suspension was filtered and washed with pyridine (5 mL). Thefiltrate was concentrated in vacuo. The resulting mixture was stirred inHCl (30 mL, 1N), ethyl acetate (15 mL) and ether (15 mL) for 2 hrs. Thesuspension was filtered and washed with water (30 mL) and ethyl acetate(30 mL) to give a dark brown solid, which was stirred with methanol (50mL) at room temperature overnight. The suspension was filtered andwashed with methanol to give3-(2-methyl-5-nitro-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione as ablack solid (490 mg, 27% yield). The solid was used in the next stepwithout further purification.

A mixture of3-(2-methyl-5-nitro-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione (250mg) and Pd(OH)₂ on carbon (110 mg) in DMF (40 mL) was shaken underhydrogen (50 psi) for 12 hrs. The suspension was filtered through a padof Celite and washed with DMF (10 mL). The filtrate was concentrated invacuo and the resulting oil was purified by flash column chromatography(silica gel, methanol/methylene chloride) to give3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione as awhite solid (156 mg, 69% yield): HPLC: Waters Symmetry C₁₈, 5 μm,3.9×150 mm, 1 mL/min, 240 nm, 10/90 CH₃CN/0.1% H₃PO₄, 3.52 min (99.9%);mp: 293-295° C.; ¹H NMR (DMSO-d₆) δ 2.10-2.17 (m, 1H, CHH), 2.53 (s, 3H,CH₃), 2.59-2.69 (m, 2H, CH₂), 2.76-2.89 (m, 1H, CHH), 5.14 (dd, J=6, 11Hz, 1H, NCH), 6.56 (d, J=8 Hz, 1H, Ar), 6.59 (d, J=8 Hz, 1H, Ar), 7.02(s, 2H, NH₂), 7.36 (t, J=8 Hz, 1H, Ar), 10.98 (s, 1H, NH); ¹³C NMR(DMSO-d₆) δ 20.98, 23.14, 30.52, 55.92, 104.15, 110.48, 111.37, 134.92,148.17, 150.55, 153.62, 162.59, 169.65, 172.57; LCMS: MH=287; Anal.Calcd. for C₁₄H₁₄N₄O₃+0.3H₂O: C, 57.65; H, 5.05; N, 19.21. Found: C,57.50; H, 4.73; N, 19.00.

6.4 Identification of Direct Compound Targets

To identify the direct target of thalidomide and other related drugs, wedevelop an affinity purification technique. A compound control(“Compound A”) coupled to Affigel-10 (10 μmol drug per mg Affigel) wasused for affinity purification experiments.

Compound A (TNF IC₅₀=622 nM; Jurkat IL-2 EC₂₀₀=3.47 μM)

Proteins were isolated from Jurkat T cell lysates by binding to theCompound A-Affigel followed by elution using free Compound A. Coomassiestained bands were excised and sent to Harvard Microchemistry Facilityfor sequence analysis. The proteins were proteolytically digested andanalyzed by microcapillary reverse-phase HPLC nano-electrospray tandemmass spectrometry on a Finnigan LCQ DECA quadrupole ion trap massspectrometer. The MS/MS spectra were correlated with known sequencesusing the algorithm Sequest and other programs, then the peptidesequences were reviewed by a scientist for consensus with known proteinsand the results manually confirmed for fidelity.

Summary of Results:

DDB1 (DNA damage binding protein 1 (XPCE, human) was affinity purifiedfrom Jurkat extracts using Compound A-immobilized beads. DDB1 was highlyrepresented in the eluted fractions (108 peptides) compare with forexample with the second proteins most represented Glycogen branchingenzyme (55 peptides). DDB1 directly interacts with CRBN and could hadbeen pull-down by virtue of its binding to CRBN.

6.5 CRBN siRNAs Knocked Down CRBN in Sensitive Multiple Myeloma CellLines

The role of cereblon (CRBN) in lenalidomide MOA was confirmed usingknock-down experiments in two lenalidomide sensitive multiple myelomacell lines, H929 and U266B1. It was found that down regulation of CRBNabrogates drug-induced cell cycle arrest, activation of tumorsuppressors, inhibition of oncogenes and global changes in geneexpression profiles and ubiquitination.

10 different single and pool siRNAs against CRBN were evaluated in H929and U266B1 cells. RT-PCR was used to test the knockdown efficiency after24 and 48 hour transfection. The results showed that the CRBN-siRNA-1,CRBN-siRNA-7, CRBN-siRNA-9, CRBN-siRNA-10, and CRBN-siRNA-11significantly reduced the expression of CRBN mRNA as compared with othersiRNAs and mock siRNA (Table 1).

TABLE 1 Effect of CRBN-siRNAs on CRBN gene expression % inhibition ofCRBN mRNA relative to CRBN mRNA levels in Mock control siRNA conc siRNA10 nM 25 nM 50 nM Invitrogen siRNAs Low GC Neg ctrl  88/100 85.5/77.585/77 CRBN-7 22/25   14/17.5 16.5/17   CRBN-8 47.5/61   29.5/41    33/37.5 CRBN-9 67.5/66.4 61.5/52   42/37 Dharmacon siRNAs Smart poolneg ctrl 87/78 72.5/84   98.5/88.5 CRBN Smartpool   28/34.5 19.3/28.518/17 CRBN-9 55/40 28.5/33   17.5/23   CRBN-10 23/35 20.5/29     20/22.5J-021086-11 22/27 20/17 16.3/15.3 J-021086-12 55/53 37/39 21/25

6.6 Knockdown of CRBN Abrogates the Anti-Proliferative Effect of Drugsin Multiple Myeloma Cells

Drugs such as lenalidomide and pomalidomide have directanti-proliferative activity against MM cells by inducing cell cyclearrest in G1 phase, followed by a decrease in viability. To study therole of CRBN in lenalidomide and other drugs anti-proliferativeactivities, H929 and U266B1 cells, two sensitive myeloma lines, weretransfected with CRBN-siRNAs or control siRNAs for 24, 48, 72 and 96hours. Cells were treated 24 h after transfection with DMSO (0.1%),pomalidomide (1 μM) and lenalidomide (10 μM) for 1, 2, 3 days toevaluated the compound effect on CRBN protein, mRNA expression and theeffect on cell cycle and proliferation. RT-PCR and western blot assaysshowed that CRBN siRNAs knocked down CRBN. CRBN down regulation wasconfirmed by RT-PCR and Western blot using the CRBN70 antibody (FIG. 1).Treatment with lenalidomide and pomalidomide affected neither mRNA norprotein expression of CRBN significantly.

Lenalidomide and pomalidomide induced a delay of cell cycle progression,measured as the decrease of the number of cells in S phase, of 40% and50% respectively in control mock and negative control siRNA-transfectedcells (average inhibition of 3 days of treatment) (FIGS. 2A & 2B).Knockdown of CRBN markedly abrogated the lenalidomide andpomalidomide-induced delay in cell cycle progression in U266B1 cells.Id.

The effect of CRBN in H929 cells was also evaluated. H929 cells weretransfected with mock, negative control siRNA and CRBN-siRNA-7 for 24,48, 72 and 96 h. Cells were treated 24 h after transfection with DMSO(0.1%), pomalidomide (1 μM), lenalidomide (10 μM) or3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione(“Compound B”) for 1, 2, 3 days and the effect on cell cycle andproliferation investigated. Lenalidomide, pomalidomide and Compound Binduced a delay of cell cycle progression, measured as the decrease ofthe number of cells in S phase, in control mock and negative controlsiRNA-transfected cells after 72 h treatment (FIG. 2C). Knockdown ofCRBN markedly abrogated immunomodulatory drugs-induced delay in cellcycle progression in H929 cells (FIG. 2C) from of 50 to 4% forlenalidomide, from 70 to 15% for pomalidomide, and from 65 to 22% forCompound B.

6.7 Knockdown of CRBN Abrogated Effect of Drugs on Cell Cycle, TumorSuppressor and Apoptotic Proteins

To further investigate the effects of CRBN on the cell cycle arrestinduced by drugs we used RT-PCR and Western blot analysis to measure thelevels of key cell cycle and apoptotic regulators. U266B1 cells weretransfected with mock, negative control siRNA, CRBN-siRNA-7 andCRBN-siRNA-11 for 24, 48, 72 and 96 h (FIG. 3A). Cells were treated 24 hafter transfection with DMSO (0.1%), pomalidomide (1 μM) andlenalidomide (10 μM) for 1, 2, 3 days and the effect on mRNA and proteinlevels of the cyclin-dependent kinase (CDK) inhibitor p21^(WAF-1) wereevaluated. It has been shown that lenalidomide- and pomalidomide-inducedcell cycle arrest is dependent on up-regulation of p21^(WAF-1) in U2669.Our results showed that p21 was up-regulated by lenalidomide andpomalidomide in control mock and negative control siRNA-transfectedcells (FIG. 3C) indicating a decrease of S phase cells resulted fromlenalidomide and pomalidomide G1 arrest (FIG. 3B). However, knockdown ofCRBN prevents the induction of p21^(WAF-1) indicating the successfulabrogation of G1 arrest and renewal of S phase progression (FIGS. 3C &3D).

In H929 cells, the cell cycle arrest in G1 phase by the drugs coincideswith a reduction of tumor suppressor, pRb, phosphorylation and theoncogene and myeloma survival factor IRF4. Western blot analysis showedthat lenalidomide, pomalidomide and Compound B decreased phosphorylationof pRB (FIGS. 4A & 4B) and total level of protein IRF4 (FIGS. 4C & 4D).The effect of the drugs was reduced by knockdown of CRBN suggesting thatinhibition of cell cycle progression by the drugs requires CRBN protein.

6.8 Knockdown of CRBN Inhibits Lenalidomide and Pomalidomide Effects onGene Expression

Gene expression profile using microarray technology identified 759 or609 genes differentially modulated in negative control siRNA-transfectedU266B1 cells treated with pomalidomide (1 μM) or lenalidomide (10 μM)compared to DMSO treated control (±1.7 fold; P<0.05 ANOVA p-value)(FIGS. 5A & 5B).

Representative significant Gene Ontology (GO) classes of down regulatedgenes in pomalidomide include: cell cycle (87 genes; P=5.2E-56), mitosis(43 genes; P=1.2E-39), cytoskeleton (58 genes; P=2.6E-16) and responseto DNA damage stimulus (36 genes; P=1.8E-21). Representative significantGene Ontology (GO) classes of up regulated genes include: antigenprocessing and presentation (12 genes; P=2.8E-17), immune response (35genes; P=5.70E-14) and cell death (50 genes; P=1.7E-10).

Representative significant Gene Ontology (GO) classes of up regulatedgenes in lenalidomide include: antigen processing and presentation (11genes; P=1.9E-16), immune response (37 genes; P=3.9E-13) and cell death(55 genes; P=1.0E-10) (Tables 4 and 5). Lenalidomide and pomalidomideeffects on cell cycle and gene expression profiles in U266 wereabrogated by knockdown of CRBN using 4 different siRNAs (FIGS. 5A-5C).

6.9 CRBN Levels are Decreased in Multiple Myeloma Cells Resistant toLenalidomide or Pomalidomide

Although the development of lenalidomide-based therapies has improvedsignificantly clinical responses, most multiple myeloma patientseventually relapse or become refractory to their therapeutic regimens.Multiple myeloma cell lines resistant to the anti-proliferative effectsof lenalidomide have been developed in order to evaluate the potentialmechanisms responsible for lenalidomide resistance.

Due to the relevance of CRBN for the anti-proliferative response oflenalidomide, pomalidomide and other immunomodulatory the levels of CRBNprotein was compared in matched pairs of parental sensitive lines withacquired lenalidomide- or pomalidomide-resistance cells. Consistent withCRBN being required for the anti-proliferative activity of lenalidomideand pomalidomide, the levels of CRBN protein were significantly lower inthe pomalidomide-resistant cells line DF15R and thelenalidomide-resistant cells, H929 R10-1, H929 R10-2, H929 R10-3, H929R10-4 and MM1/R compared to the matched parental lines (Table 2).

TABLE 2 Fold difference of CRBN protein level between lenalidomide- orpomalidomide-resistant cells and the matched parental cells CRBN proteinfold decrease relative to matched parental lines DF15R 7.16E−07H929R10-1 0.503989 H929R10-2 0.295482 H929R10-3 0.459599 H929R10-40.659341 MM1/R 0.172249

6.10 Genomic DNA Sequencing of CRBN, DDB1 and GSK3 from Human Cell Linesand Primary Cells

The target genes CRBN, DDB1, and GSK3B were sequenced from a variety ofhuman cell lines and primary cells. Sequencing of the CRBN geneidentified mutations in lenalidomide resistant human cell lines whichare absent in parental cells.

Sequence enrichment, NexGen sequencing, and data analysis yielded theresults shown in Tables 3-6 below. Each table lists the cell lines fromwhich the data are derived. The reference nucleotide refers to the wildtype nucleotide at that position. “Coverage” refers to the total numberof reads at that position. The overall mutation score (“Score”) is basedon the concept of Phred scores with a maximum value of 30, meaning theprobability that the mutations call is wrong is 1/1000; 20, 1/100; and10, 1/10. A Score, however, does not necessarily mean that the mutationis more than likely a false mutation. A low Score implies only that themutation cannot be called a true mutation with absolute certainty.“Mutation Call” denotes the nucleotide change. For example, T>TGindicates a heterozygous change from reference T to T and G. T>Gindicates a homozygous change from reference T to G. The term “c.”refers to the coding sequence while “IVS” refers to an intron variant.The “Amino Acid Change” column uses similar notation to the “MutationCall” column.

Results

In the large-scale sequence analysis of 27 cell lines, more intronicmutations were found than exonic mutations. The multiple myeloma cellline, ANBL-6, showed a mutation in CRBN of the lenalidomide-resistantline that was not seen in the wild type. ANBL-6.wt was treated only withDMSO and the CRBN coding sequence exactly matched the reference.However, in the ANBL-6.R10R which has resistance to 10 μM lenalidomide,a SNP in the coding region c.745C>CA caused an amino acid change 249D>YDin the protein. This amino acid change mapped to the DDB1 binding domainof CRBN.

A silent mutation found in the CRBN coding region of HepG2 and JeKo-1was heterozygous c.735A>AG with KMS-12-BM as homozygous c.735A>G. Thecell line, OPM2, had a different silent mutation in the coding region ofCRBN, c.1209C>CT. No amino acid changes resulted from these SNPs.

The sequencing results for DDB1 uncovered a SNP in the coding regionc.909T>TA which resulted in an amino acid change 303E>ED in bothANBL-6.wt and ANBL-6.R10R cell lines. A different SNP was found in theJurkat cell line, with a mutation c.2143C>CT in the DDB1 coding regionthat changed the amino acid sequence 715V>VI.

A silent mutation found in the DDB1 coding region of ANBL-6.wt,ANBL-6.R10R, and SH-SY5Y at c.153G>GA did not change the amino acidsequence. A sample of PBMC from a healthy donor also had a silentmutation in the DDB1 coding region c.2265G>GA that did not change theamino acid sequence.

One GSK3β mutation was found in the PMBC sample. The mutation in thecoding region c.1187C>CT resulted in an amino acid change 396R>RQ. Thismutation did not map to the kinase domain. Other mutations are shown inTable 11. Most of these have low coverage. This could be due to primerstargeting these regions and/or sequencing error.

While no consistent mutations in CRBN, DDB1 and GSK3-β were found tocorrelate with resistance to lenalidomide or pomalidomide in these MMcell lines, sporadic mutations such as the 249D>YD CRBN mutationobserved in lenalidomide-resistant ANBL-6 MM cells exemplify the type ofpolymorphism which might occur in such genes within the clinicalsetting, and which might constitute a mechanism of resistance. Furtherstudies would be needed to confirm or refute this hypothesis.

TABLE 3 CRBN Mutations Amino Acid Cell line Mutation Call Change OPM2c.1209C > CT 403T > TT^(a) HepG2 c.735A > AG 245Y > YY^(a) JeKo-1c.735A > AG 245Y > YY^(a) KMS-12-BM c.735A > G 245Y > Y^(a) ANBL-6.R10Rc.745C > AC 249D > YD^(b) KMS-12-BM IVS1148 + 21_1148 + 22insA SH-SY5YIVS1148 + 21_1148 + 22insA SKMM2 IVS1148 + 21_1148 + 22insA JurkatIVS1148 + 23delA EJM IVS1329 + 18_1329 + 19insAACT H929_R10-4 IVS1329 +18_1329 + 19insAACT JJN-3 IVS1329 + 18_1329 + 19insAACT OPM2 IVS1329 +18_1329 + 19insAACT SH-SY5Y IVS1329 + 18_1329 + 19insAACT MM.1S.R10RIVS175 − 9A > AG MM.1S.wt IVS175 − 9A > AG PBMC IVS175 − 9A > AG HepG2IVS175 − 9A > AG, IVS1148 + 21_1148 + 22insA ANBL-6.wt IVS175 − 9A > GEJM IVS175 − 9A > G H929_D1 IVS175 − 9A > G H929_R10-1 IVS175 − 9A > GH929_R10-2 IVS175 − 9A > G H929_R10-3 IVS175 − 9A > G H929_R10-4 IVS175− 9A > G H929-1uM- IVS175 − 9A > G CC5013 JeKo-1 IVS175 − 9A > G JJN-3IVS175 − 9A > G Jurkat IVS175 − 9A > G KMS-12-BM IVS175 − 9A > G OPM2IVS175 − 9A > G RPMI-8226 IVS175 − 9A > G SH-SY5Y IVS175 − 9A > GSK-Hep1 IVS175 − 9A > G SKMM2 IVS175 − 9A > G U266_B1 IVS175 − 9A > GANBL-6.R10R IVS175 − 9A > G, IVS1148 + 21_1148 + 22insA U87 IVS175 −9A > G, IVS1148 + 21_1148 + 22insA, IVS528 − 29_528 − 28insCT H929IVS175 − 9A > G, IVS1329 + 18_1329 + 19insAACT H929-0.1uM- IVS175 − 9A >G, IVS1329 + 18_1329 + 19insAACT CC4047 H929-DMSO IVS175 − 9A > G,IVS528 − 29_528 − 28insCT KMS-12-BM IVS528 − 29_528 − 28insCT SKMM2IVS528 − 29_528 − 28insCT U266_B1 IVS528 − 29_528 − 28insCT ^(a)= silentmutation; ^(b)= region required for DDB1 interaction

TABLE 4 DDB1 Mutations Amino Acid Cell line Mutation Call ChangeANBL-6.R10R c.153G > AG 51P > PP^(a) ANBL-6.wt c.153G > AG 51P > PP^(a)SH-SY5Y c.153G > AG 51P > PP^(a) Jurkat c.2143C > CT 715V > VI PBMCc.2265G > AG 755S > SS^(a) ANBL-6.R10R c.909T > AT 303E > DE ANBL-6.wtc.909T > AT 303E > DE ANBL-6.wt IVS1123 − 29A > G EJM IVS1123 − 29A > GH929 IVS1123 − 29A > G H929_R10-3 IVS1123 − 29A > G H929-1uM- IVS1123 −29A > G CC5013 HepG2 IVS1123 − 29A > G JeKo-1 IVS1123 − 29A > G MM.1S.wtIVS1123 − 29A > G OPM2 IVS1123 − 29A > G SK-Hep1 IVS1123 − 29A > GU266_B1 IVS1123 − 29A > G ANBL-6.wt IVS1123 − 30C > T EJM IVS1123 −30C > T H929 IVS1123 − 30C > T H929_R10-3 IVS1123 − 30C > T H929-1uM-IVS1123 − 30C > T CC5013 HepG2 IVS1123 − 30C > T JeKo-1 IVS1123 − 30C >T MM.1S.wt IVS1123 − 30C > T OPM2 IVS1123 − 30C > T SK-Hep1 IVS1123 −30C > T U266_B1 IVS1123 − 30C > T PBMC IVS1225 + 30T > C MM.1S.R10RIVS1225 + 30T > CT MM.1S.wt IVS1225 + 30T > CT RPMI-8226 IVS1225 + 30T >CT JJN-3 IVS1862 − 26A > AT OPM2 IVS1862 − 26A > AT H929-0.1uM- IVS1862− 27A > AC CC4047 JJN-3 IVS1862 − 27A > AC OPM2 IVS1862 − 27A > ACSH-SY5Y IVS1862 − 27A > AC H929 IVS2278 − 26T > GT H929-1uM- IVS2278 −26T > GT CC5013 Jurkat IVS2278 − 26T > GT KMS-12-BM IVS2278 − 26T > GTOPM2 IVS2278 − 26T > GT ANBL-6.wt IVS2278 − 27C > CT EJM IVS2278 − 27C >CT H929 IVS2278 − 27C > CT H929_R10-2 IVS2278 − 27C > CT H929-1uM-IVS2278 − 27C > CT CC5013 JJN-3 IVS2278 − 27C > CT Jurkat IVS2278 −27C > CT KMS-12-BM IVS2278 − 27C > CT OPM2 IVS2278 − 27C > CT PBMCIVS2278 − 27C > CT SH-SY5Y IVS2278 − 27C > CT SK-Hep1 IVS2278 − 27C > CTSKMM2 IVS2278 − 27C > CT U266_B1 IVS2278 − 27C > CT U87 IVS2278 − 27C >CT ANBL-6.wt IVS2278 − 28C > T H929 IVS2278 − 28C > T H929_R10-2 IVS2278− 28C > T H929_R10-3 IVS2278 − 28C > T H929-1uM- IVS2278 − 28C > TCC5013 Jurkat IVS2278 − 28C > T KMS-12-BM IVS2278 − 28C > T OPM2 IVS2278− 28C > T PBMC IVS2278 − 28C > T SH-SY5Y IVS2278 − 28C > T SKMM2 IVS2278− 28C > T U266_B1 IVS2278 − 28C > T U87 IVS2278 − 28C > T JJN-3IVS2661 + 6C > CT PBMC IVS2832 + 6C > CT RPMI-8226 IVS2832 + 6C > CT^(a)= silent mutation

TABLE 5 GSK3β Mutations Amino Acid Cell line Mutation Call Change PBMCc.1187C > CT 396R > RQ Jurkat IVS282 + 3delT U266_B1 IVS366 + 29A > AGH929 IVS366 + 29A > G Jurkat IVS909 + 11delA

TABLE 6 Other Mutations Chr Y: LOC100288025 Chr M: ATP6, COX1, CYTB,ND1, ND4, ND4L, ND5 Chr 2: C2orf67 Chr 1: ITLN1 Chr 19: KLK10 Chr 17:KRT15 Chr 14: PRMT5, FMNL1 Chr 9: NOTCH1

6.11 Study of the Relationship Between Drugs and the UbiquitinProteasome System

Specific antibodies for poly-ubiquitin chains were used to study theeffect of immunomodulatory drugs on global levels of ubiquitination.H929 cells were treated with lenalidomide (1 μM), pomalidomide (1 μM) orproteasome inhibitor MG132. After 30 minutes cells were process forimmunofluorescence. Global levels of K63-linked polyubiquitination werequantified by Cellomics. As shown in FIGS. 7A & 7B, immunomodulatorydrugs decrease total K48-linked polyubiquitination but not K-63-linkedubiquitination in H929.

6.12 Study of the Effect of Drugs on Global Changes of Ubiquination andProtein Abundance

The effects of lenalidomide and pomalidomide on protein ubiquinationwere investigated using CST Ubiscan technology. Treated samples weresent to Cell Signaling Technology for ubiquinated peptide enrichment andquantification by LC/MS/MS. Raw intensity was used for this analysis toidentify peptides that are significantly regulated by the drugs, or thedrugs with proteasome inhibitor MG132. Analysis showed that comparedwith MG132, the effects of lenalidomide and pomalidomide onubiquitinated peptides are small. In the MG132 combo, more ubiquinatedpeptides are observed (compared with their controls). 162 uniqueubiquitinated peptides were significantly up-regulated by lenalidomideand pomalidomide alone, or with MG132 at 1 hour or 4 hours. Thesepeptides correspond to 176 unique proteins. Top few groups are:nucleosome, chromatin, protein-DNA complex assembly, Histone H2A. Amongthe 176 proteins, we found five proteins that belong to“ubiquitin-protein ligase activity” category, they are MDM2, HERC2,UBE2D3 (only by pomalidomide), UBE2N (lenalidomide only), UBE2M (both).Results for hits categorized by conditions are shown in FIGS. 8 & 9(without MG132) and FIGS. 10 & 11 (with MG132).

6.13 CRBN Knockdown Effect on Drug Induced TNFα and IL-2 in PrimaryT-Cells

In these studies, human T cells were isolated from blood and treatedwith 1 μg/ml PHA-L at 37° C. After 24 hr stimulation T cells weresubjected to siRNA transfection with the indicated siRNAs. Knockdownefficiencies were analyzed by qRT-PCR after 24 h transfections and theremaining transfected cells seeded in 96-well plate s pre-bound withOKT3 and treated with DMSO or 1 and 10 μM thalidomide, lenalidomide,pomalidomide and phthalimide in duplicate at 37° C. for 48 hours. After48 hours, the supernatants were collected and tested for TNFα □ and IL-2production by ELISA (FIGS. 12A-12D). This data indicates that siRNAknockdown of CRBN abrogates drug-induced TNFα and IL-2 production inanti-CD3-stimulated primary human T cells.

6.14 Knocking Down Cul4A and Cul4B Together Partially AbrogatesLenalidomide, Pomalidomide, or Compound B-Induced TNFα and IL-2Induction in T Cells

Cul4A knockdown efficiency prior to drug treatment was measured. Cul4Agene expression was knocked down by 82% and 76% by Cul4A siRNA-1 andCul4A+Cul4B siRNA, respectively (FIG. 13A). Cul4B gene expression wassuppressed by 70% and 63% byCul4B and Cul4A+Cul4B siRNA, respectively.The individual knock down of Cul4A or Cul4B had no effect on T cellTNF-α or IL-2 production induced by 10 μM lenalidomide, pomalidomide orCompound B (FIGS. 13B & C). However, the double knockdown of Cul4A andCul4B together resulted in a significant but partial reversal of thecompound-induced elevation of TNF-production due to lenalidomide,pomalidomide, and Compound B (FIG. 13B). There was a trend of a reversalof IL-2 induction when Cul4A and Cul4B were knocked down (FIG. 13C).These data suggest that lenalidomide, pomalidomide, and CompoundB-mediated T cell costiulation is dependent on the expression of Cul4Aand Cul4B, and that these proteins serve redundant functions in the Tcell.

6.15 CRBN Expression and Sensitivity to Lenalidomide in Lymphoma Cells

The antiproliferative activity of lenalidomide versus baseline CRBNexpression was studied in diffuse large B-cell lymphoma (DLBCL) celllines. The following DLBCL cell lines were evaluated for sensitivity tolenalidomide: OCI-Ly10-NCI, U2932, OCI-Ly-3, DB, RIVA, TMD8, Toledo,OCI-Ly-19, Pfeiffer, WSU-DLCL2, Karpas-1106P and SU-DHL-4. Results areshown in FIG. 14.

6.16 Preparation of CRBN-DDB1 Complex

Primers were designed for cloning of CRBN into pBV-ZZ-HT-LIC andpBV-notag-LIC. Two primers were prepared, “CRBN_For” (SEQ ID NO: 14) andCRBN_Rev.” (SEQ ID NO: 15)

CRBN_For: GTGCCGCGTGGCTCCATGATGGCCGGCGAAGGAGATCACRBN_Rev: GCTTCCTTTCGGGCTTATTACAAGCAAAGTATTACTT

The primers were used to amplify the CRBN gene from a cDNA library. Theproduct was gel purified and treated with T4 DNA polymerase in thepresence of TTP only to make single-stranded ends compatible forligation independent cloning. The CRBN DNA was then annealed topBV-ZZ-HT-LIC to create CRBN_034 (FIG. 15A).

For DDB1 two primers were prepared, “DDB1_For” and “DDB1_Rev.”

DDB1_For:  (SEQ ID NO: 2)TCGGGCGCGGCTCTCGGTCCGAAAAGGATGTCGTACAACTACGTGGTAAC DDB1_Rev:(SEQ ID NO: 3) GCTTCCTTTCGGGCTTATTTTTCGAACTGCGGGTGGCTCCAATGGATCCGAGTTAGCTCCT

The DDB1_Rev adds a StrepTag at the C-termini of DDB1. The DDB1 gene wasamplified from a cDNA library, gel purified, and treated with T4polymerase in the presence of TTP. The DDB1 gene was then annealed topBV-notag-LIC to create the plasmid DDB1_004 (FIG. 15B).

Expression of Constructs in Baculovirus and Purification

The constructs could were tested for expression in insect cells.Recombinants pBV-HT-LIC and pBV-GST-LIC plasmids were transformed intoDH10Bac to produced bacmids. Integrity of the recombination was followedby a blue-white screen and PCR. The recombinant bacmids were used totransfect 9×105 Sf9 adherent cells per well in serum free-Grace media.After the transfection, fresh Grace media containing antibiotics andglutamine was added.

Infection was followed by observation of the cell monolayers undermicroscope. After 5 to 7 days, the supernatants were saved (P1 virus)and the pellets were analyzed for recombinant protein expression. Virusamplification was in 24 deep well plates with 4 ml of 2×106 Sf9 cells/mlper well. Aliquots were removed to assess the kinetics of proteinexpression. After 4 days, the plate was centrifuged, the supernatantwere saved (P2 virus) and the pellets were analyzed by mini scalepurification of the tagged proteins. The viruses were finally amplifiedin 750 ml of Grace media containing antibiotics and glutamine and thesupernatants saved as P3 virus. Pellets were saved and purified usingeither the AKATxpress or the AKTA purifier from Amersham Biosciences.

Purification

Cell pastes containing CRBN-DDB1 were lysed in a buffer containing 50 mMTris pH 8.0, 500 mM NaCl, 20 mM Imidazole, 10% Glycerol, and 2 mM DTTand protease inhibitors. The lysate was then cleared by centrifugation.The tagged proteins were then purified from the supernatants using theAKTA Express from Amersham Biosciences. Five ml HisTrap HP columns wereused for the affinity step while a 16 mm×60 cm Sephacryl S-200 HR wasused for the size exclusion steps. After loading the column, was columnwas washed with 20 volumes of lysis buffer, then 10 column volumes of 50mM Tris pH 8.0, 1000 mM NaCl, 40 mM Imidazole, 10% Glycerol, and 2 mMDTT. Elution of the bound proteins was performed with lysis buffercontaining 500 mM Imidazole. Eluted proteins were directly injected intothe gel filtration column equilibrated in 25 mM Tris pH 8.0, 200 mMNaCl, 5% Glycerol, and 2 mM DTT. Fractions were analyzed by 4-20% SDSpolyacrylamide gel electrophoresis.

Fractions containing both CRBN and DDB1 were pooled and digested withThrombin to remove the ZZ-HT tag from CRBN. Digestion was carried at 4°C. for 5-6 hours with 1:2000 (weight/weight) of thrombin and CRBN-DDB1.Cleaved CRBN-DDB1 was diluted in 25 mM Tris pH 8.0, 5% Glycerol, and 2mM DTT, and loaded onto an 8 ml MonoQ column (Amersham-Pharmacia)equilibrated in 25 mM Tris pH 8.0, 75 mM NaCL, 5% Glycerol, and 2 mMDTT. After loading the column was wash with 2 volumes of 25 mM Tris pH8.0, 75 mM NaCl, 5% Glycerol and 2 mM DTT, and the bound proteins wereeluted with a gradient from 75 mM to 400 mM in 25 mM Tris pH 8.0, 5%Glycerol and 2 mM DTT

A final gel filtration was done to polish the CRBN-DDB1 complex. Thefraction pooled from the MonoQ were loaded onto a 140 ml S200HR Gelfiltration column and ran in 25 mM Tris pH 8.0, 200 mM NaC, 5% Glycerol,and 2 mM DTT. Fractions were analyzed and positive fractions were pooledand concentrated to approximately 15 mg/ml. Aliquots were stored at −80°C.

6.17 Ubiscan Ubiquitination Experiments

The results of 1 hour and 4 hour ubiquitination experiments are shown inFIGS. 17-22, which demonstrate that certain peptides are regulated bylenalidomide and/or pomalidomide.

The tables of FIG. 23 show Ubiscan data results for lenalidomide,pomalidomide and Compound B. Ub-proteins IKZF3, RPL19, PCM1 and NEDD8were commonly increased in abundance by Rev and Pom in U266 and CompoundB in T cells. Proteins GNB2L1 and HNRNPR were commonly decreased inabundance by Rev and Pom in U266 and Compound B in T cells.

Treatment of T cells with Compound B resulted in a greater abundance oftwo ubiquinated peptides (vs. DMSO control), SECTM1 and ZC3H15.

Proteins in common with lenalidomide, pomalidomide and Compound B:IKZF3, RPL19, PCM1, NEDD8, GNB2L1, and HNRNPR.

TABLE 7 Final Ubiscan hits for lenalidomide and pomalidomide (1 hr) SEQID ID Gene Name Peptide Sequence NO: 1 147 G3BP2 QYYTLLNK*APEYLHR 16 2904 COPS6 QVCEIIESPLFLK*LNPM#TK 17 3 905 COPS6 QVCEIIESPLFLKLNPM#TK* 185 1137 VCPIP1 VGDVQGQESESQLPTKIILTGQK* 19 9 1582 HYOU1 FFGDSAASM#AIK*NPK20 10 2541 MCM7 FLQEFYQDDELGK*K 21 13 3043 CCT3 SM#M#K*MLLDPMGGIVM#TNDGN22 AILR 14 3226 LMNB2 LSSDQNDK*AASAAR 278 23 16 3910 C12orf51LSPYLEDVSGGMWPVVHIQK*KNTK 24 18 3992 EDEM3 ATGDPYYLEVGK*TLIENLNK 25 254982 RAB28 VVK*ADIVNYNQEPM#SR 26 33 7093 HNRNPUL1IGWSLDSCSTQLGEEPFSYGYGGT 27 GK*K 35 7556 ABCF2 YGLTGK*QQVSPIR 28 37 7740DNAJC1 ALPHLIQDAGQFYAKYK* 29 38 7798 GNAS VLTSGIFETKFQVDK*VNFHM#FD 30VGGQRDER 39 8193 SLC16A1 ASLEK*AGK 31 42 10418 RPL19 HMGIGK*R 32 4310556 RPL36 AMELLKVSK* 33 44 10565 RPL4 MFAPTK*TWR 34 46 11831 UBE2Q1ELK*LLESIFHR 35 47 12009 ARMC6 NLVAHGQAFSK*PILDLGAEALI 36 M#QAR 50 12698KLHL7 ISVNSNNVQSLLDAANQYQIEPV 37 K*K 51 12891 NUP37 FCTSAADMK*IR 38 5212942 POC5 VVTSAQQK*AGR 39 54 13152 SNRPE IM#LK*GDNITLLQSVSN 40

TABLE 8 Final Ubiscan hits for lenalidomide and pomalidomide (MG 1 hr)SEQ ID ID Gene Name Peptide Sequence NO: 1 105 DOK5 K*ASSKGPK  41 2 324SHCBP1 AYQDYILADCK*ASEVQEFTA  42 EFLEK 4 389 YWHAE MK*GDYHR  43 5 512FERMT3 ETTLSYYK*SQDEAPGDPIQQ  44 LNLK 6 904 COPS6 QVCEIIESPLFLK*LNPM#TK 45 7 905 COPS6 QVCEIIESPLFLKLNPM#TK*  46 8 1010 PCM1 LMAAK*QK  47 91112 SMC4 SVAVNPK*EIASK  48 10 1114 TOPORS K*IQEQDIINFR  49 11 1323HSPA1A SAVEDEGLK*GK  50 12 1323 HSPA1B SAVEDEGLK*GK  50 13 1452 HSPA8DISENK*R  51 14 1623 ATRX DNRGGIKSK*  52 15 1679 DUTIFYPEIEEVQALDDTERGSGG  53 FGSTGK*N 16 1773 H2AFJ K*GNYAER  54 17 1773HIST1H2AA 17 18 1773 HIST1H2AC K*GNYAER  54 19 1773 HIST1H2AI K*GNYAER 54 20 1773 HIST1H2AK K*GNYAER  54 21 1773 HIST2H2AA3 K*GNYAER  54 221773 HIST2H2AB K*GNYAER  54 23 1773 HIST2H2AC K*GNYAER  54 24 1863 H2AFXK*TSATVGPK  55 25 3146 LMNA TLEGELHDLRGQVAK*LEAAL  56 GEAK 26 3192 LMNAK*LESTESR  57 27 3377 TUBA1A DVNAAIATIK*TK  58 28 3377 TUBA1BDVNAAIATIK*TK  58 29 3377 TUBA1C DVNAAIATIK*TK  58 30 3377 TUBA3CDVNAAIATIK*TK  58 31 3377 TUBA3D DVNAAIATIK*TK  58 32 3377 TUBA3EDVNAAIATIK*TK  58 33 3963 DHFR LTEQPELANK*VDM#VWIVGG  59 SSVYK 34 3984DHX15 EVDDLGPEVGDIK*IIPLYST  60 LPPQQQQR 37 4111 GBA LLLPHWAK*VVLTDPEAAK 61 41 4199 IDH3G NTGK*SIANK  62 43 4471 PPAT CGLPYVEVLCK*NR  63 44 4938GNB3 GQQK*TV  64 45 4938 H2AFZ GQQK*TV  64 46 5121 VAV1 VLK*YHLLLQELVK 65 47 5121 VAV3 VLK*YHLLLQELVK  65 48 5126 VAV1 IDGELK*ITSVER  66 495252 UCK2 VLTSEQKAK*  67 50 5377 ALDOA ADDGRPFPQVIK*SK  68 54 5582 SDHAAFGGQSLKFGK*  69 59 6046 PSMA7 AITVFSPDGHLFQVEYAQEA  70 VK*K 60 6046PSMA8 AITVFSPDGHLFQVEYAQEA  70 VK*K 61 6049 PSMA7 AITVFSPDGHLFQVEYAQEA 71 VKK* 62 6049 PSMA8 AITVFSPDGHLFQVEYAQEA  71 VKK* 64 6567 IRAK2CPIPAFPDSVK*PEKPLAAS  72 VR 65 6662 PRKDC NILEESLCELVAKQLK*  73 66 6662LOC731751 NILEESLCELVAKQLK*  73 67 6741 PRKDC GHDEREHPFLVK*GGEDLR  74 686741 LOC731751 GHDEREHPFLVK*GGEDLR  74 70 7093 HNRNPUL1IGWSLDSCSTQLGEEPFSYG  75 YGGTGK*K 72 7306 PCBP2 LVVPASQCGSLIGK*GGCK  7675 7542 ABCE1 VAETANEEEVKK*  77 78 7783 GNAL VLAGK*SK  78 79 7783 GNASVLAGK*SK  78 80 7939 KCNAB2 IGVGAM#TWSPLACGIVSGK*  79 YDSGIPPYSR 81 8156PTCH1 DKPIDISQLTK*QR  80 82 8173 SEMA4A VCK*NDVGGEK  81 84 8478 ASCC3FQALQDNCK*K  82 85 8779 HMGB2 IK*SEHPGLSIGDTAK  83 86 8808 IFI16KK*EVDATSPAPSTSSTVK  84 88 8973 ILF2 K*ILGQEGDASYLASEISTWD  85 GVIVTPSEK89 9023 IRF4 QWLIDQIDSGK*YPGLVWENE  86 EK 91 9082 NACANILFVITKPDVYK*SPASDTY  87 IVFGEAK 92 9465 SUPT5H SSVGETVYGGSDELSDDITQQ 88 QLLPGVK*DPNLWTVK 93 9525 TRIP4 GK*DVEFPNDYPSGCLLGCVD  89 LIDCLSQK 949764 EEF1A1 IGYNPDTVAFVPISGWNGDNM  90 #LEPSANM#PWFK*GWK 95 9764 eEF1AL3IGYNPDTVAFVPISGWNGDNM  90 #LEPSANM#PWFK*GWK 96 9766 EEF1A1IGYNPDTVAFVPISGWNGDNM  91 #LEPSANM#PWFKGWK* 97 9766 eEF1AL3IGYNPDTVAFVPISGWNGDNM  91 #LEPSANM#PWFKGWK* 98 9794 EEF1A1 AAGAGK*VTK 92 99 9794 eEF1AL3 AAGAGK*VTK  92 106 10407 RPL19 TLSK*EEETKK  93 10710409 RPL19 TLSKEEETK*K  94 108 10411 RPL19 TLSKEEETKK*  95 111 11082RPS25 LVSK*HR  96 112 11397 COPS3 LKAM#DQEITVNPQFVQK*SM  97#GSQEDDSGNKPSSYS 113 11426 CUL9 ILK*AHGEK  98 115 11794 UBAP2LIDLAVLLGK*TPSTMENDSSNL  99 DPSQAPSLAQPLVFSNSK 116 11931 ADRM1 GTTVTPDK*R100 120 12423 FAM10A4 AK*SEENTKEEKPDSK 101 121 12423 FAM10A5AK*SEENTKEEKPDSK 101 122 12423 ST13 AK*SEENTKEEKPDSK 101 123 12447FAM129A VLK*QYDYDSSTIR 102 125 12609 IGJ FVYHLSDLCK*K 103 126 12627IKZF3 SHTVEKPYK*CEFCGR 104 131 13060 S100A6 EGDKHTLSK*K 105 132 13212SSR2 KYDTPK*TK 106 133 13250 SSX1 SK*AFDDIATYFSK 107 134 13306 TIPRLLK*VVPTTDHIDTEKLK 108 136 13598 AMBRA1 REPFAVVK*TASEM#ER 109 137 13676COPG ALQQYTLEPSEKPFDLK*SV 110 PLATAPM#AEQR 138 13280 TBCCGK*DAASSTKVDAAPGIPPA 111 VESIQDSPLPK 139 13281 TBCC GKDAASSTK*VDAAPGIPPA112 VESIQDSPLPK

TABLE 9 Final Ubiscan hits for lenalidomide and pomalidomide (4 hr) SEQID ID Gene Name Peptide Sequence NO: 1 236 GNB2L1 LK*TNHIGHTGYLNTVTVSPDG113 SLCASGGK 2 487 CTNNB1 LLHPPSHWPLIK*ATVGLIR 114 3 512 FERMT3ETTLSYYK*SQDEAPGDPIQQL  44 NLK 5 663 PKP2 TYDMLK*AGTTATYEGR 115 6 732HSP90B1 NLGTIAK*SGTSEFLNK 116 9 1112 SMC4 SVAVNPK*EIASK  48 10 1863H2AFX K*TSATVGPK  55 11 2508 HIST2H2AB KTESHKPGK*NK 117 12 2512HIST2H2AB KTESHKPGKNK* 118 13 2579 NAP1L2 GLIGYVLDTDFVESLPVKVK* 119 153712 TPD52 SFEEK*VENLK 120 16 3742 VAPA RYCVRPNSGIIDPGSTVTVSV 121M#LQPFDYDPNEKSK* 17 3799 AKR1B1 LLLNNGAK*M#PILGLGTWK 122 18 3916 CHPFSALTAHPVRDPVHMYQLHK*A 123 FAR 19 3955 DDX24 ATNEGLSLM#LIGPEDVINFK 124 K*20 4035 FASN DGLLENQTPEFFQDVCKPK* 125 23 4181 HMOX1 K*AALEQDLAFWYGPR 12625 4347 NANS QLLPCEMACNEK*LGK 127 27 4938 GNB3 GQQK*TV  64 28 4938 H2AFZGQQK*TV  64 29 4968 NET1 IGEATK*PDGTVEQIGHILVS 128 WLPR 31 5672 ANP32EKLELSDNIISGGLEVLAEK*C 129 PNLTYLNLSGNK 32 5676 CDC25CSLNQYPALYYPELYILK*GGY 130 R 33 5732 PPP1CB AK*YQYGGLNSGRPVTPPR 131 356543 CHEK1 M#CGTLPYVAPELLK*R 132 36 6546 CSNK1A1 LFLIDFGLAK*K 133 376546 CSNK1AlL LFLIDFGLAK*K 133 38 6563 IRAK1 GTLAYLPEEYIK*TGR 134 406843 PRKAG2 KK*DVSSPGGSGGK*K*NAS 135 QK* 41 6858 CPSF3LVNCYM#PANGETVTLPTSPS 136 IPVGISLGLLK*R 42 7344 RARS SDGGYTYDTSDLAAIK*QR137 43 7672 C7orf42 QSNPEFCPEK*VALAEA 138 44 7717 CPNE1 SEVIK*NNLNPTWK139 46 8137 NUP88 IYSLREPQTPTNVIILSEAE 140 EESLVLNK*GR 47 8331 TCIRG1QEENK*AGLLDLPDASVNGW 141 SSDEEK 48 8723 FAF1 K*SPM#M#PENAENEGDALL 142QFTAEFSSR 49 8734 FUBP3 ITGDAFK*VQQAR 143 54 9253 PSMC3LAGPQLVQM#FIGDGAK*LV 144 R 55 9526 TSG101 ASLISAVSDK*LR 145 56 9794EEF1A1 AAGAGK*VTK  92 57 9794 eEF1AL3 AAGAGK*VTK  92 58 9933 EIF2S1ADIEVACYGYEGIDAVK*EA 146 LR 62 10690 RPL9 K*FLDGIYVSEK 147 64 11010 RPS2AEDK*EWMPVTK 148 65 11010 LOC645018 AEDK*EWMPVTK 148 66 11585 NAE1CINITK*QTPSFWILAR 149 67 11831 UBE2Q1 ELK*LLESIFHR 35 68 12110 C13orf40KPDLRIIEQEEK* 150 70 12242 COMMD4 LEVAAAPGTPAQPVAM#SLS 151 ADK*FQVLLAELK71 12320 DDIT4 GALLDVCVEQGK*SCHSVGQ 152 LALDPSLVPTF 72 12458 FAM60ATPVFSFLDLTYWK*R 153 73 12545 HLA-E GYEQFAYDGK*DYLTLNEDLR 154 74 12758MAGEA3 AREPVTK*AEM#LGSVVGNW 155 QYFFPVIFSK 75 12758 MAGEA6AREPVTK*AEM#LGSVVGNW 155 QYFFPVIFSK 76 12865 NHP2 IK*ADPDGPEAQAEACSGER156 78 13290 TBL3 LWTIK*NNECVR 157 79 13930 NEDD8 QMNDEK*TAADYK 158 80403 YWHAZ YLAEVAAGDDK*K 159 81 805 SHISA5 SQPPYNPAYM#DAPK*AAL 160 822089 HIST1H1A GTLVQTK*GTGASGSFK 161 83 2089 HIST1H1B GTLVQTK*GTGASGSFK161 84 2089 HIST1H1C GTLVQTK*GTGASGSFK 161 85 2089 HIST1H1DGTLVQTK*GTGASGSFK 161 86 2089 HIST1H1E GTLVQTK*GTGASGSFK 161 87 13902NEDD8 LIYSGK*QMNDEK 162 88 14057 RPS27A K*IQDK*EGIPPDQQR 163 89 14057RPS27AP5 K*IQDK*EGIPPDQQR 163 90 14057 UBA52 K*IQDK*EGIPPDQQR 163 9114057 UBB K*IQDK*EGIPPDQQR 163 92 14057 UBC K*IQDK*EGIPPDQQR 163

TABLE 10 Final Ubiscan hits for lenalidomide and pomalidomide (MG 4 hr)SEQ ID ID Gene Name Peptide Site NO: 1 11 PLAA FIIDNTK*GQM#LGLGNPSFSDP164 FTGGGR 2 259 HGS ACGQIFCGK*CSSK 165 3 260 HGS ACGQIFCGKCSSK* 166 4358 UBE2M VGQGYPHDPPK*VK 167 5 361 UBE2M VGQGYPHDPPKVK* 168 6 533 IL32GDK*EELTPQK 169 7 826 ANXA7 AM#K*GFGTDEQAIVDVVANR 170 8 917 CP110NKMLGTSSKESEELLK*SK* 171 9 1112 SMC4 SVAVNPK*EIASK  48 10 1146 ANP32BIFGGLDM#LAEKLPNLTHLNLSG 172 NKLK* 11 2579 NAP1L2 GLIGYVLDTDFVESLPVKVK*119 12 2591 PURA FFFDVGSNK*YGVFM#R 173 13 2768 VCPKAFEEAEK*NAPAIIFIDELDAI 174 APKR 14 2791 VCP ELQELVQYPVEHPDKFLK* 175 152929 ACTB DIK*EKLCYVALDFEQEMATAAS 176 SSSLEK 16 2929 ACTG1DIK*EKLCYVALDFEQEMATAAS 176 SSSLEK 17 2929 POTEE DIK*EKLCYVALDFEQEMATAAS176 SSSLEK 18 3231 LSP1 QEM#LLSLK*PSEAPELDEDEGF 177 GDWSQRPEQR 19 3281NES TSLSFQDPK*LELQFPR 178 20 3575 AUP1 FPSSGPVTPQPTALTFAK*SSWAR 179 213588 MAN1A1 GYAWGLNELK*PISK 180 22 3589 MAN1A1 K*GSGPAALR 181 23 3712TPD52 SFEEK*VENLK 120 24 3740 VAPA YCVRPNSGIIDPGSTVTVSVM#L 182QPFDYDPNEK*SK 27 4483 PPIA VSFELFADK*VPK 183 29 4655 TPI1ELASQPDVDGFLVGGASLK*PEF 184 VDIINAKQ 30 4656 TPI1KQSLGELIGTLNAAK*VPADTEV 185 VCAPPTAYIDFAR 31 4938 GNB3 GQQK*TV  64 324938 H2AFZ GQQK*TV  64 33 5308 ALDOA VDK*GVVPLAGTNGETTTQGLDG 186 LSER 345459 MTX1 FTGAPLK*VHKISNPWQSPSGTL 187 PALR 35 5573 PTRH2TQIAPGSQTVLGIGPGPADLIDK 188 VTGHLK*LY 36 5640 MYL6 VFDK*EGNGTVMGAEIR 18937 5792 PPP3CA HLTEYFTFK*QECK 190 38 5792 PPP3CB HLTEYFTFK*QECK 190 395810 PPP3CC SQATGFPSLITIFSAPNYLDVY 191 NNK*AAVLK 40 6427 UCHL5TLAEHQQLIPLVEKAK* 192 41 6449 USP15 GPSTPNVK*NSNYCLPSYTAYK 193 42 6558HUNK K*PEPHQPGPGSTGIPHK*EDP 194 LMLDM#VR 45 6861 CPSF6KTTQSGQMSGEGK*AGPPGGSSR 195 46 7088 HNRNPR IK*ALLER 196 47 7088 SYNCRIPIK*ALLER 196 48 7186 MORC3 STNQQTATDVSTSSNIEESVNH 197 M#DGESLK*LR 497240 PABPC1 VVCDENGSK*GYGFVHFETQEA 198 AER 50 7522 ABCE1 STALK*ILAGK 19951 7672 C7orf42 QSNPEFCPEK*VALAEA 138 52 8030 MLC1 K*GSMSDSANILDEVPFPAR200 53 8239 SLC35F2 TAEPAESSVPPVTSIGIDNLGL 201 K*LEENLQETHSAVL 54 8370TMEM57 KHNLGINNNNILQPVDSKIQEI 202 EYM#ENHINSK* 56 9215 POLR2LCFTCGKIVGNK*WEAYLGLLQA 203 EYTEGDALDALGLKR 57 9354 RSF1AQIDPVLLK*NSSQQDNSSR 204 58 9398 SP140 MK*ESPGSQQCCQESEVLER 205 59 9468TBL1XR1 DK*LAQQQAAAAAAAAAAASQQ 206 GSAK 61 10524 RPL30KSEIEYYAM#LAK*TGVHHYSG 207 NNIELGTACGK*YYR 62 11010 RPS2 AEDK*EWMPVTK148 63 1101 LOC645018 AEDK*EWMPVTK 148 64 11435 DCUN1D1QFM#IFTQSSEK*TAVSCLSQN 208 DWK 65 11510 MDM2 ENWLPEDKGKDKGEISEK* 209 6611580 MIB1 SSEDATDDISSGNIPVLQK*DK 210 DNTNVNADVQK 67 11581 MIB1SSEDATDDISSGNIPVLQKDK* 211 DNTNVNADVQK 68 11830 UBE2OSTDSQCGTVIDVNIDCAVK*LI 212 GTNCIIYPVNSK 69 11969 ANKRD13A LTLDLM#KPK*213 71 12110 C13orf40 KPDLRIIEQEEK* 150 72 12127 C19orf43QKTEDEVLTSK*GDAWAK 214 73 12129 C19orf43 QKTEDEVLTSKGDAWAK* 215 74 12171CAPN8 LAGKDSEITANALK* 216 75 12259 COPS8 K*PVAGALDVSFNKFIPLSEPA 217PVPPIPNEQQLAR 76 12322 DDIT4 K*LYSSEQLLIEEC 218 77 12432 FAM114A1SVLTGGLDALEFIGK* 219 78 12564 HSPBP1 AMQQQVQK*LK 220 79 12929 PLIN2GAVTGSVEK*TK 221 80 12930 PLIN2 GAVTGSVEKTK* 222 81 13264 STRBPM#VLLPVM#K*FPTYPVPHYSFF 223 82 13561 WDR6 MVK*VDPETR 224 83 13849 VAMP8NLQSEVEGVK*NIMTQNVER 225 84 2097 HIST1H1A K*ALAAAGYDVEKNNSR 226 85 2097HIST1H1C K*ALAAAGYDVEKNNSR 226 86 2097 HIST1H1D K*ALAAAGYDVEKNNSR 226 872097 HIST1H1E K*ALAAAGYDVEKNNSR 226 88 2097 HIST1H1T K*ALAAAGYDVEKNNSR226 89 3242 MYO18A INSLQDMVTK*YQKR 227 90 4677 UAP1 LTLSK*AGQEHLLR 22891 8262 SLC3A2 IK*VAEDEAEAAAAAK 229

6.18 Efficacy of Lenalidomide in Activated B-Cell Like Subtype DLBCL isDependent Upon Expression of IRF4 and CRBN Cell Proliferation Assay

Cell proliferation was assessed using the ³H-thymidine incorporationassay. Briefly, logarithmically growing DLBCL cells were cultured in96-well culture plates in complete media with the indicatedconcentration of lenalidomide or DMSO control. Following incubation at37° C. for 5 days, 1 μCi ³H-thymidine (GE Healthcare Biosciences,Piscataway, N.J.) was added to each well for the final 5 hours ofincubation. Cells were then harvested onto UniFilter GF/C filter plates(PerkinElmer, Waltham, Mass.) using a cell harvester (Tomtec, Hamden,Conn.) and the plates were allowed to dry overnight. The ³H-thymidineincorporation of each well was then measured using a TopCount NXTMicroplate Scintillation and Luminescence Counter (Packard BioScience,Meriden, Conn.). The percent inhibition of cell proliferation wascalculated and normalized to DMSO control.

Protein Expression Analysis

Cells were treated with test compounds or 0.1% DMSO for indicated times.Following incubation, cells were collected, pelleted withcentrifugation, and immediately lysed in 0.1 ml lysis buffer containing10 mM Tris-HCl pH 8.0, 10 mM EDTA, 150 mM NaCl, 1% NP-40, 0.5% SDS, 1 mMDTT, 1 mM Na₃VO₄, plus Complete protease inhibitor cocktail (RocheApplied Science, Indianapolis, Ind.), then processed with a Qiashredder™(Qiagen, Valencia, Calif.) for 1 minute and frozen on dry ice. Sampleswere diluted with 6×SDS sample buffer and then boiled for 5 min.Approximately 30 μl of this mixture was loaded per lane on a CriterionPrecast 4-12% Tris-HCl gel (Bio-Rad, Hercules, Calif.), electrophoresed,and transferred to nitrocellulose membranes (Bio-Rad, Hercules, Calif.).The membranes were blocked for 1 hour at room temperature using blockingbuffer (LI-COR Biosciences, Lincoln, Nebr.), then incubated overnight at4° C. with antibodies against either BCL-10, IRF4, CRBN or β-actin.Membranes were washed and incubated with IRDye Secondary Antibodies(1:30,000) for 1 hour at room temperature. A standard protocol was thenfollowed for signal detection, using the Odyssey® Infrared ImagingSystem and software (LI-COR Biosciences, Lincoln, Nebr.).

NF-κB Activity Assays

Logarithmically growing DLBCL cells were treated with test agents asindicated. Nuclear extracts were prepared using a Nuclear Extract Kit(Active Motif, Carlsbad, Calif.) and protein concentration wasdetermined by the bicinchoninic acid assay (Thermo Scientific, Rockford,Ill.). Detection of NF-κB activity was performed using a sensitiveoligo-based colorimetric enzyme-linked immunosorbent assay (ELISA)method, according to the instructions of the manufacturer (ActiveMotif). Briefly, nuclear extracts of DLBCL cells were hybridized to96-well plates coated with wild-type DNA oligonucleotides containing asingle copy of the NF-κB consensus binding sequence. Bound NF-κB proteinwas then detected with antibodies specific for p50, p65 (Rel A) or p70subunits. A secondary antibody conjugated to horseradish peroxidase wasadded and plates were then read by spectrophotometry at 450 nm with areference wavelength of 655 nm. NF-κB activity was calculated based onOD450/655 nm.

For the NF-κB-driven luciferase reporter gene assay, cells weretransfected with the pGL4.32 [luc2P/NF-κB-RE/Hygro] plasmid (Promega,Madison, Wis.) using Nucleofector kit V and program 013 according to themanufacturer's protocol (Amaxa Biosystems, Gaithersburg, Md.). After 24hours of transfection, cells were treated with test agents for 2 daysand lysates were prepared using the Dual-Glo® Luciferase Assay System(Promega). Luciferase activities of each sample were measured using aTopCount NXT Microplate Scintillation and Luminescence Counter (PackardBioScience).

Real-Time Quantitative Reverse Transcriptase-PCR Analysis

After 48 hours of cell treatment, total RNA was purified with RNeasy®Mini Kits using QiaCube™ system (Qiagen Inc., Valencia, Calif.).Real-time quantitative RT-PCR with 25-100 ng of total RNA was performedusing the reverse transcription kit and Taqman® PCR probes specific forthe genes of interest according to standard methods (Applied BiosystemsInc.). The quantity of product was normalized toglyceraldehyde-3-phosphate dehydrogenase as the endogenous housekeepinggene. Fold increase of gene expression was calculated using comparativeCt method (2^(−ΔΔCt)).

Electroporation for Overexpression and Knockdown of IRF4

To knockdown IRF4, cells were transfected with Silencer® Select siRNA(small interfering RNA, Applied Biosystems) directed against IRF4, CRBN,or Silencer® Select Negative Control siRNAs at a final concentration of0.2-1 μM using Nucleofector® Kit V for transfection. For IRF4overexpression, cells were transfected with IRF4- or green fluorescentprotein (GFP)-cytomegalovirus (CMV) expression plasmids (OriGeneTechnologies, Rockville, Md.) for 24 h using the Cell Line Nucleofectorkit V as above-mentioned. After 24 hours of transfection, cells weretreated with lenalidomide for 2 days before luciferase assay, RT-PCRgene expression analysis and Western blot.

Human Tumor Xenograft Model

Female CB17 severe combined immunodeficiency (SCID) mice (6-12 weeksold) were obtained from the Charles River Laboratory (Wilmington, Mass.)and maintained in microisolator cages under sterile conditions. A totalof 10×10⁶ OCI-Ly10 DLBCL cells in 100% Matrigel (Becton Dickinson, SanJose, Calif.) were injected subcutaneously into the right flank of mice.Mice were monitored 2 or 3 times a week for the appearance of tumors.Once the tumors reached an average size of 100-150 mg, 10 mice in eachgroup were treated with either vehicle (0.5% carboxymethyl cellulose:0.25% Tween 80 in deionized H₂O) or indicated doses of lenalidomide(qd×28, p.o.) or the positive control vincristine (q4d×4, i.v.). Micewere monitored daily for health status as well as tumor growth. Tumorsof all mice were measured with a digital caliper and volumes calculatedwith the following formula: tumor volume (mm³)=length (mm)×width (mm)².Mice were killed when tumor size exceeded 1000 mm³.

Statistical Analysis

Analyses for multiple group comparisons were performed with one-wayanalysis of variance, followed by Dunnett's post-test, and correlationanalyses were carried out using the two-tailed P-value Pearson testusing GraphPad Prism® version 5.01 (San Diego, Calif.). A value ofP<0.05 was considered significant in all analyses.

Results 6.18.1 ABC-DLBCL Cells are More Sensitive to Lenalidomide thanNon-ABC-DLBCL Cells

In order to define the place of lenalidomide for DLBCL therapy inspecific patient populations and to understand the molecular mechanismsof efficacy, a panel of DLBCL cell lines was collected in this study.The DLBCL subtypes of the cell lines were confirmed based on literatureinformation (Lenz G, et al., Proc Natl Acad Sci USA 2008; 105: 13520-5)and molecular analysis, including intracellular NF-κB activity or IRF4expression, as well as gene expression profiling of key signature genesof activated B cells. See FIGS. 24A-24C. Cell proliferation of DLBCLcell lines was found to be inhibited to various degrees by treatmentwith lenalidomide at a concentration range of 0.01-100 μM. Lenalidomidehad minimal effects on proliferation of PBML and GCB-DLBCL cells, butsignificantly inhibited proliferation of ABC-DLBCL cell lines, exceptfor OCI-Ly3. See FIG. 24. Lenalidomide treatment also induced apoptosisof sensitive cell lines such as OCI-Ly10. See FIG. 26.

6.18.2 Lenalidomide Reduces IRF4 Expression in ABC-DLBCL Cells

To understand the molecular mechanism of lenalidomide on ABC-DLBCLcells, the effects of lenalidomide on IRF4 expression in these cellswere investigated. Lenalidomide treatment for 1-3 days was found tosignificantly downregulate IRF4 protein levels in sensitive cell linessuch as U2932 and OCI-Ly10, but not the insensitive line OCI-Ly3 cells.See FIGS. 27A-C. Lenalidomide-induced decrease of IRF4 expressionoccurred as early as 1 day of drug treatment, with similar kinetics tothat of the inhibitors (zVRPR-fmk and LY-333,531, respectively) of MALT1and PKCβ, two key enzymes involved in NF-κB activation upon BCRengagement in B cells. In OCI-Ly3 cells, neither lenalidomide nor thePKCβ inhibitor had any appreciable effect on IRF4 levels during 1-3 daytreatments. However, the MALT1 inhibitor suppressed IRF4 expression inOCI-Ly3 cells, with complete inhibition observed after 2-3 days oftreatment. Taken together, these data suggest that lenalidomide-mediatedinhibition of IRF4 expression may be an important mechanism and appearsto be related to cell sensitivity to the drug.

6.18.3 Lenalidomide Reduces CARD11-BCL-10-MALT1 Complex Activity ofABC-DLBCL Cells

Our DNA sequencing data confirmed previous reports (Lenz, G., et al.,Science 2008, 319: 1676-9) that the lenalidomide-insensitive ABC-DLBCLline OCI-Ly3 has a unique point mutation in CARD11 within the exonsencoding the coiled-coil domain, while lenalidomide-sensitive ABC-DLBCLlines OCI-Ly10, U2932, TMD8, and Riva do not. See FIG. 28. This mutationhas been reported to cause constitutive formation and activation ofCARD11-BCL-10-MALT1 (CBM) complex of BCR signaling pathway, leading toNF-κB overactivation in lymphoma cells. See Lenz, G., et al., Science2008, 319: 1676-9; Thome, M. et al., Cold Spring Harb Perspect Biol.2010; 2: a003004. To investigate the potential involvement of CBMcomplex in lenalidomide-induced IRF4 inhibition in these cells, theeffect of lenalidomide on the complex activity was examined by measuringMALT1 paracaspase enzymatic activity. MALT1 is activated uponassociation with BCL-10 and CARD11 to form active CBM complex and thencleaves its binding partners, such as BCL-10. See FIGS. 29A-C.

Similar to the effect of the specific MALT1 inhibitor and PKCβinhibitor, lenalidomide inhibited MALT1-induced BCL-10 cleavage in aconcentration-dependent manner in the sensitive ABC-DLBCL cell linesOCI-Ly10 and U2932. Time-kinetic studies revealed that while MALT1- andPKCβ-inhibitors affected BCL-10 cleavage within 1 day of treatment,significant inhibition of BCL-10 cleavage by lenalidomide occurred after2 days of treatment. See FIGS. 29A and 29B. Unlike the MALT1 inhibitor,neither the PKCβ inhibitor nor lenalidomide had any effect on BCL-10cleavage in OCI-Ly3 cells, presumably due to CARD11 mutation whichcauses over-activation of CBM complex. See FIG. 29C. These data suggestthat lenalidomide, similar to the PKCβ inhibitor, can significantlyblock CBM complex formation/activation or other upstream events insensitive ABC-DLBCL cells.

6.18.4 Lenalidomide Reduces NF-κB Activity of ABC-DLBCL Cells but NotNon-ABC Subtype Cells

The effect of lenalidomide on NF-κB activity in various DLBCL cells wasexamined by measuring the level of NF-κB subunit proteins binding toconsensus sequences and the NF-κB-driven luciferase activity. Asexpected, NF-κB in ABC-DLBCL cells demonstrated increased NF-κB DNAbinding compared to non-ABC-DLBCL. See FIG. 24B. An NF-κB-drivenluciferase assay demonstrated that lenalidomide inhibitedtranscriptional activity of NF-κB by 32-56% in thelenalidomide-sensitive ABC-DLBCL cell lines OCI-Ly10 and U2932 after 2day drug treatment. See FIG. 30A. Lenalidomide also partially inhibitedDNA binding by Rel A/p65, p50 and c-rel/p70 NF-κB subunits in aconcentration-dependent manner in several ABC-DLBCL cell lines, althoughthe effect was not as potent as the control IKKα/β inhibitor CC-415501.See FIGS. 30B and 30C. In contrast, lenalidomide had no effect on NF-κBDNA binding in GCB-DLBCL lines nor in normal peripheral bloodmononuclear cells. The lenalidomide-insensitive ABC-DLBCL OCI-Ly3 linecontaining the CARD11 mutation showed significant NF-κB inhibition onlyby the IKKα/β inhibitor, and not by lenalidomide. See FIGS. 30B and 30C.These data suggest that lenalidomide inhibits NF-κB signaling at CBMcomplex or upstream events in sensitive ABC-DLBCL cells.

6.18.5 Alteration of IRF4 Expression in Sensitive ABC-DLBCL CellsConfers Resistance to Lenalidomide

The dependency role of IRF4 expression in BCR-NF-κB signal transductionand the sequent effects of lenalidomide treatment upon IRF4 wereinvestigated. Lenalidomide-sensitive ABC-DLBCL cells were transfectedwith IRF4-specific siRNA or IRF4-CMV-based expression plasmid tomodulate IRF4 expression. When U2932 or OCI-Ly10 cells were transfectedwith IRF4 siRNA to knock down the expression of IRF4, NF-κBtranscriptional activity was diminished by 36-53%. See FIG. 31A. ThusIRF4 siRNA mimicked the effects of lenalidomide on NF-κB in these cells.The addition of lenalidomide to the IRF4 siRNA-transfected cells led toeven further downregulation of NF-κB transcriptional activity.

In contrast to IRF4 siRNA transfection, over-expressing IRF4 in thesecells for 24 h significantly increased NF-κB activity by 3.6-7.9 fold.See FIG. 31B. Furthermore, IRF4 overexpression decreased proteinexpression levels of the CBM-component BCL-10. See FIG. 31C. Inaddition, IRF4 overexpression antagonized the effect of lenalidomide andthe PKCβ inhibitor but not the IKK inhibitor on NF-κB-driven luciferaseexpression in both U2932 and OCI-Ly10 cells. See FIG. 31D. Therefore,the positive feedback effect of IRF4 on the BCR-NF-κB pathway appearedto be at a point somewhere between PKCβ and IKK.

6.18.6 Cereblon is Required for Lenalidomide Effect on ABC-DLBCL Cells

Cereblon has been shown to be a primary mediator of the teratogenicityof thalidomide. Due to its critical role in the anti-myeloma andimmunomodulatory activities of lenalidomide and pomalidomide, the roleof cereblon in lenalidomide sensitivity of DLBCL was investigated. InABC-DLBCL cells, knockdown of CRBN with siRNA conferred resistance tolenalidomide as demonstrated by the abrogation of the inhibitory effectsof lenalidomide on IRF4 expression, BCL-10 cleavage, NF-κB activity andproliferation of these cells, while the activity of inhibitors to PKCβand IKK remained unaffected. See FIGS. 32A-32D. These data indicate thatantitumor effects of lenalidomide on ABC-DLBCL cells require thepresence of cereblon.

6.18.7 Lenalidomide Down-Regulates IRF4/NF-κB Signaling in OCI-Ly10Mouse Xenograft Model

To confirm the relevance of lenalidomide-mediated inhibition ofNF-κB/IRF4 signaling in vivo in ABC-DLBCL, a subcutaneous OCI-Ly10xenograft model was established. Lenalidomide at 3-30 mg/kg (po, qdX28)significantly decreased tumor size in this model (p<0.01). See FIG. 33A.No pronounced toxicity of lenalidomide was observed based upon mouseweight throughout study. Compared with vehicle control group,lenalidomide treatment for 7 days reduced IRF4 expression and BCL-10cleavage by 15-35% (p<0.05). See FIG. 33B. These data demonstrate thatlenalidomide can reduce IRF4 expression in vivo and delays tumor growthin an ABC-DLBCL model, supporting the potential value of lenalidomide asa therapeutic in the treatment of ABC-DLBCL in clinical studies.

6.18.8 IRF4 and CRBN Baseline mRNA Levels and “ABC Scores” of DLBCLCells Correlate with Sensitivity to Lenalidomide

As multiple studies have demonstrated greater lenalidomide sensitivityof ABC-DLBCL cells versus non-ABC-subtypes, potential biomarkerspredictive for a therapeutic response to lenalidomide were studied.Pooled in vitro data from eleven DLBCL cell lines of various subtypesshowed that lenalidomide sensitivity of DLBCL subtypes highly correlatedto the baseline level of IRF4 and CRBN mRNA expression. Additionally,the overall “ABC score,” calculated based on baseline levels ofsignature genes of ABC-DLBCL cells proposed by Staudt, et al. (Ann. Rev.Med., 2002, 53: 303-18), correlated to lenalidomide activity and furtherconfirms the unique sensitivity of this subtype. See FIGS. 34A and 25.Lenalidomide-sensitive ABC-DLBCL cell lines tended to express higherCRBN and IRF4 protein levels than GCB-DLBCL cell lines. See FIG. 34B.Notably, the lenalidomide-resistant OCI-Ly3 ABC-DLBCL cell line wasdevoid of CRBN protein expression. These data support the preferentialefficacy of lenalidomide in ABC-DLBCL seen in clinical studies, andsuggest that the “ABC score,” or the expression of IRF4 or CRBN itself,may serve as potential biomarkers for the prediction of lenalidomideefficacy.

6.19 Polyclonal CRBN70 Antibody

Rabbit polyclonal antibody CRBN70 was generated by inoculating rabbitswith the CRBN peptide sequence EEFHGRTLHDDD (residues 1-12 of SEQ IDNO:1), wherein a C-terminal cysteine EEFHGRTLHDDDC (SEQ ID NO: 1)(underlined) is further used to couple the peptide to Keyhole LimpetHemocyanin (KLH).

Seq. Mod. EEFHGRTLHDDDC-KLH KLH-Peptide Cys

The peptide depicted in SEQ ID NO:1 used to make the antibodycorresponds to amino acids 65-76 (bolded) of CRBN isoform 1 (NP_057386)(SEQ ID NO:12):

1 magegdqqda ahnmgnhlpl lpaeseeede mevedqdske akkpniinfd tslptshtyl 61gadmeefhgr tlhdddscqv ipvlpqvmmi lipgqtlplq lfhpqevsmv rnliqkdrtf 121avlaysnvqe reaqfgttae iyayreeqdf gieivkvkai grqrfkvlel rtqsdgiqqa 181kvqilpecvl pstmsavqle slnkcqifps kpvsredqcs ykwwqkyqkr kfhcanitsw 241prwlyslyda etlmdrikkq lrewdenlkd dslpsnpidf syrvaaclpi ddvlriqllk 301igsaiqrlrc eldimnkcts lcckqcqete ittkneifsl slcgpmaayv nphgyvhetl 361tvykacnlnl igrpstehsw fpgyawtvaq ckicashigw kftatkkdms pqkfwgltrs 421allptipdte deispdkvil cl

It is noted that isoform 2 of CRBN (GenBank Accession No. NP_001166953;SEQ ID NO:13) uses an alternate in frame splice site that results in theelimination of the alanine (underlined), but has no other changes:

1 magegdqqda ahnmgnhlpl lpeseeedem evedqdskea kkpniinfdt slptshtylg 61admeefhgrt lhdddscqvi pvlpqvmmil ipgqtlplql fhpqevsmvr nliqkdrtfa 121vlaysnvqer eaqfgttaei yayreeqdfg ieivkvkaig rqrfkvlelr tqsdgiqqak 181vqilpecvlp stmsavqles lnkcqifpsk pvsredqcsy kwwqkyqkrk fhcanltswp 241rwlyslydae tlmdrikkql rewdenlkdd slpsnpidfs yrvaaclpid dvlriqllki 301gsaiqrlrce ldimnkctsl cckqcqetei ttkneifsls lcgpmaayvn phgyvhetlt 361vykacnlnli grpstehswf pgyawtvaqc kicashigwk ftatkkdmsp qkfwgltrsa 421llptipdted eispdkvilc l

The polyclonal CRBN70 antibody was purified. Purified antibody was thentitered by indirect ELISA against the peptide or protein bound to asolid-phase to measure the reactivity of the antibodies after elutionand the amount of antibody remaining in serum (the flow-through).

Volume Conc. Total Titer Flow- Sample (mL) (mg/mL) (mg) (ng) throughCRBN70 6 0.414 2.484 5 2048

The eluent “titer” indicates the minimum concentration at which theCRBN70 antibody can effectively detect the CRBN antigen. The“flow-through” titer represents the reactivity of the antibodiesremaining in the serum after it has been passed through the column.

6.20 Sequences of VH and VL of Anti-CRBN Antibodies

Rabbits were primed with amino acid sequence 65-76 (SEQ ID NO:1) ofhuman CRBN (SEQ ID NO:12), and the spleen was removed for IgG subtypingand monoclonal antibody creation.

The IgG heavy and light chains from two CGN-6 hybridoma clones weresequenced by Epitomics. The two clones were CGN-6-1-11 and CGN-6-4-5.

Brief Methods Description of RabMAb IgG Molecular Cloning:

Messenger RNA (mRNA) from hybridoma cells was isolated usingTURBOCAPTURE Kit (Qiagen: Catalog #72232) following the manufacturer'ssuggested protocol, and then reverse transcribed into cDNA usingoligo-dT primer. The variable region of heavy chain (VH) was PCRamplified using proprietary primers OYZ64-2 and OYZvh3. The entire lightchain (LC) was PCR amplified using proprietary primers OYZ62 and OYZ71.The PCR products were resolved on 1% agarose gel followed bypurification using Qiagen gel purification kit (Qiagen: Catalog #28704),and the purified DNA fragments were subjected to sequencing.

CGN-6-1-11- Heavy chain nucleotide sequence (SEQ ID NO: 4):ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCACTGTCAGTCAGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGTACAGTCTCTGGATTCTCCCTCAGTTACTATGGAGTGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTAGAATACATCGGATACATTTATAGTGATAGTGATAAGACATACTACGCGACCTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCGACCACGGTGGATTTGAAAATCACCAGTCCGACAATCGAGGACACGGCCACCTATTTCTGTGCCAGAGGTACTCCGCTTGCTAGTTATAGCATCTGGGGCCCAGGCACCCTGGTCACCGTCTCCTTAGGGCAACCTAAGGCTCCATCAGTCTTCCCACTGGCCCCCTGCTGCGGGGACACACCCAGCTCCACGGTGACCCTGGGCTGCCTGGTCAAAGGGTACCTCCCGGAGCCAGTGACCGTGACCTGGAACTCGGGCACCCTCACCAATGGGGTACGCACCTTCCCGTCCGTCCGGCAGTCCTCAGGCCTCTACTCGCTGAGCAGCGTGGTGAGCGTGACCTCAAGCAGCCAGCCCGTCACCTGCAACGTGGCCCACCCAGCCACCAACACCAAAGTGGACAAGACCGTTGCGCCCTCGACATGCAGCAAGCCCACGTGCCCACCCCCTGAACTCCTGGGGGGACCGTCTGTCTTCATCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCACGCACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCAGGATGACCCCGAGGTGCAGTTCACATGGTACATAAACAACGAGCAGGTGCGCACCGCCCGGCCGCCGCTACGGGAGCAGCAGTTCAACAGCACGATCCGCGTGGTCAGCACCCTCCCCATCGCGCACCAGGACTGGCTGAGGGGCAAGGAGTTCAAGTGCAAAGTCCACAACAAGGCACTCCCGGCCCCCATCGAGAAAACCATCTCCAAAGCCAGAGGGCAGCCCCTGGAGCCGAAGGTCTACACCATGGGCCCTCCCCGGGAGGAGCTGAGCAGCAGGTCGGTCAGCCTGACCTGCATGATCAACGGCTTCTACCCTTCCGACATCTCGGTGGAGTGGGAGAAGAACGGGAAGGCAGAGGACAACTACAAGACCACGCCGGCCGTGCTGGACAGCGACGGCTCCTACTTCCTCTACAGCAAGCTCTCAGTGCCCACGAGTGAGTGGCAGCGGGGCGACGTCTTCACCTGCTCCGTGATGCACGAGGCCTTGCACAACCACTACACGCAGAAGTCCATCTCCCGCTCTCCGGGTAAATGACGN-6-1-11- Heavy chain protein sequence (SEQ ID NO: 5):METGLRWLLLVAVLKGVHCQSVEESGGRLVTPGTPLTLTCTVSGFSLSYYGVSWVRQAPGKGLEYIGYIYSDSDKTYYATWAKGRFTISKTSTTVDLKITSPTIEDTATYFCARGTPLASYSIWGPGTLVTVSLGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQENSTIRVVSTLPTAHQDWLRGKEEKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK-CGN-6-1-11- Light chain nucleotide sequence (SEQ ID NO: 6):ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCACATTTGCCCAGGTGCTGACCCAGACTCCAGCCTCGGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAATTGCCAGGCCAGTCAGAGTGTTTATAAGAATAACTATTTATCCTGGTTTCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATTTACGAAGCGTCCAAACTGGCATCTGGGGTCCCCCCGCGGTTCAAAGGCAGTGGATTTGGGACACAGTTCACTTTCACCATTAGCGACCTGGAGTGTGACGATGCTGCCTTTTACTACTGTGCAGGCGGTTATTATGGTAATATTTTTTTTTTCGGCGGAGGGACCGAGGTGGTGGTCAAAGGTGATCCAGTTGCACCTACTGTCCTCATCTTCCCACCAGCTGCTGATCAGGTGGCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATGGCACCACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCACACAGTACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGGGTGACTGTTAGCGN-6-1-11- Light chain protein sequence (SEQ ID NO: 7):MDTRAPTQLLGLLLLWLPGATFAQVLTQTPASVSAAVGGTVTINCQASQSVYKNNYLSWFQQKPGQPPKLLIYEASKLASGVPPRFKGSGFGTQFTFTISDLECDDAAFYYCAGGYYGNIFFEGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC-CGN-6-4-5- Heavy chain nucleotide sequence (SEQ ID NO: 8):ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAGTGTCAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCACAGTCTCTGGATTCTCCCTCAGTAGGTATGGAGTGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAACACATCGGATACATTTATAGTGATCCTGGTATGACATTCTACGCGACCTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCGTCGACCACGGTGGATTTGAAAATGACCAGTCCGACAATCGAGGACACGGCCACCTATTTCTGTGCCAGAGGTACTCCGCTTGCTAGTTATAGCACCTGGGGCCCAGGCACCCTGGTCACCATCTCCTTAGGGCAACCTAAGGCTCCATCAGTCTTCCCACTGGCCCCCTGCTGCGGGGACACACCCAGCTCCACGGTGACCCTGGGCTGCCTGGTCAAAGGGTACCTCCCGGAGCCAGTGACCGTGACCTGGAACTCGGGCACCCTCACCAATGGGGTACGCACCTTCCCGTCCGTCCGGCAGTCCTCAGGCCTCTACTCGCTGAGCAGCGTGGTGAGCGTGACCTCAAGCAGCCAGCCCGTCACCTGCAACGTGGCCCACCCAGCCACCAACACCAAAGTGGACAAGACCGTTGCGCCCTCGACATGCAGCAAGCCCACGTGCCCACCCCCTGAACTCCTGGGGGGACCGTCTGTCTTCATCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCACGCACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCAGGATGACCCCGAGGTGCAGTTCACATGGTACATAAACAACGAGCAGGTGCGCACCGCCCGGCCGCCGCTACGGGAGCAGCAGTTCAACAGCACGATCCGCGTGGTCAGCACCCTCCCCATCGCGCACCAGGACTGGCTGAGGGGCAAGGAGTTCAAGTGCAAAGTCCACAACAAGGCACTCCCGGCCCCCATCGAGAAAACCATCTCCAAAGCCAGAGGGCAGCCCCTGGAGCCGAAGGTCTACACCATGGGCCCTCCCCGGGAGGAGCTGAGCAGCAGGTCGGTCAGCCTGACCTGCATGATCAACGGCTTCTACCCTTCCGACATCTCGGTGGAGTGGGAGAAGAACGGGAAGGCAGAGGACAACTACAAGACCACGCCGGCCGTGCTGGACAGCGACGGCTCCTACTTCCTCTACAGCAAGCTCTCAGTGCCCACGAGTGAGTGGCAGCGGGGCGACGTCTTCACCTGCTCCGTGATGCACGAGGCCTTGCACAACCACTACACGCAGAAGTCCATCTCCCGCTCTCCGGGTAAATGACGN-6-4-5- Heavy chain protein sequence (450 amino acids) (SEQ ID NO: 9):METGLRWLLLVAVLKGVQCQSVEESGGRLVTPGTPLTLTCTVSGFSLSRYGVSWVRQAPGKGLEHIGYIYSDPGMTFYATWAKGRFTISKTSSTTVDLKMTSPTIEDTATYFCARGTPLASYSTWGPGTLVTISLGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPTAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK-CGN-6-4-5- Light chain nucleotide sequence (SEQ ID NO: 10):ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCACATTTGCTCAAGTGCTGACCCAGACTCCAGCCTCCGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAATTGCCAGTCCAGTGAGAATATTTATAAGAACAACTACTTATCCTGGTTTCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTATCAGGCATCCACTCTGGCATCTGGGGTCCCATCGCGGTTCAAAGGCAGTGGATCTGGGACACGATTCAGTCTCACCATCAGCGACCTGGAGTGTGACGATGCTGCCACTTACTACTGTGCAGGCGGTTATAGTGGTAATATTTTTACTTTCGGCGGAGGGACCGAGGTGGTGGTCAAAGGTGATCCAGTTGCACCTACTGTCCTCATCTTCCCACCAGCTGCTGATCAGGTGGCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATGGCACCACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCACACAGTACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGGGTGACTGTTAGCGN-6-4-5- Light chain protein sequence (SEQ ID NO: 11):MDTRAPTQLLGLLLLWLPGATFAQVLTQTPASVSAAVGGTVTINCQSSENIYKNNYLSWFQQKPGQPPKLLIYQASTLASGVPSRFKGSGSGTRFSLTISDLECDDAATYYCAGGYSGNIFTFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC-

Amino acid sequence alignments of the heavy and light chains of theCGN6-1-11 and CGN 6-4-5 antibodies are provided in FIGS. 35 (heavychains) and 36 (light chains).

6.21 Immunoblot and Immunofluorescence with Monoclonal Anti-CRBNAntibody, CGN-6-4-5

Rabbit monoclonal antibody CGN-6-4-5 specifically recognizes thefull-length 51 kDa human CRBN protein on denaturing immunoblots.

Confocal Microscopic Immunofluorescence:

CGN-6-4-5 was diluted 1:1000. Exemplary sell staining of DF15 and DF15Rcells is shown in FIG. 37. In particular, FIGS. 37A and 37B depictconfocal immunofluorescent analysis of DF15 (left panel) and DF15R cells(right panel) using 1 μg/ml CGN-6-4-5 antibody (green) (A) or CGN-6-4-5antibody/CRBN blocking peptide mix (1:5 excess ratio) (B). Nuclearstaining was performed with Dapi (blue).

Immunoblot

CGN-6-4-5 was diluted 1:10,000 dilution in 0.1% Tween PBS buffer. Anexemplary immunoblot with myeloma cells containing endogenous CRBN(DF15), DF15R with no CRBN and HEK293 cells expressing recombinantflag-tagged CRBN is shown in FIG. 38. Peptide neutralization wasperformed by combining antibody with a five-fold (by weight) excess ofblocking peptide in 500 μl PBS and incubating with constant rotation atroom temperature for 2 hours.

6.22 Thalidomide, Lenalidomide and Pomalidomide Bind to CRBN Via theGlutarimide Moiety

The binding of pthalimide and glutarimide to CRBN was investigated inorder to elucidate the mechanism of CRBN binding of thalidomide,lenalidomide, pomalidomide and structurally similar compounds.Glutarimide bound to CRBN while pthalimide did not. Thus, these resultssupport the hypothesis that thalidomide, lenalidomide and pomalidomidebind to CRBN via the glutarimide moiety. See FIG. 39A.

6.23 CRBN Binding of Methyl-Pomalidomide is Enantioselective

The binding of S-methyl-pomalidomide and R-methyl-pomalidomide wasinvestigated in order to determine whether CRBN binding isenantioselective. S-methyl-pomalidomide had greater affinity for CRBNthan R-methyl-pomalidomide limide. See FIG. 39B.

The examples set forth above are provided to give those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the claimed embodiments, and are not intended to limit thescope of what is disclosed herein. Modifications that are obvious topersons of skill in the art are intended to be within the scope of thefollowing claims. All publications, patents, and patent applicationscited in this specification are incorporated herein by reference as ifeach such publication, patent or patent application were specificallyand individually indicated to be incorporated herein by reference.

What is claimed is:
 1. An isolated antibody or antigen binding fragmentthereof that immunospecifically binds to an epitope in CRBN, wherein theepitope has the amino acid sequence SEQ ID NO:1, and wherein theisolated antibody comprises (i) a heavy chain having complementaritydetermining regions (CDRs) consisting of the amino acids of the CDRs ofSEQ ID NO: 5 and a light chain having complementarity determiningregions (CDRs) consisting of the amino acids of the CDRs of SEQ ID NO:7; or (ii) a heavy chain having complementarity determining regions(CDRs) consisting of the amino acids of the CDRs of SEQ ID NO: 9 and alight chain having complementarity determining regions (CDRs) consistingof the amino acids of the CDRs of SEQ ID NO:
 11. 2. The isolatedantibody or antigen binding fragment thereof of claim 1, wherein theisolated antibody comprises a heavy chain having the amino acid sequencedepicted in SEQ ID NO:5 and a light chain having the amino acid sequencedepicted in SEQ ID NO:7.
 3. The isolated antibody or antigen bindingfragment thereof of claim 1, wherein the isolated antibody comprises aheavy chain having the amino acid sequence depicted in SEQ ID NO:9 and alight chain having the amino acid sequence depicted in SEQ ID NO:11. 4.The isolated antibody or antigen binding fragment thereof of claim 1,wherein the isolated antibody comprises a heavy chain variable regionconsisting of the amino acids of the heavy chain variable region of SEQID NO: 5 and a light chain variable region consisting of the amino acidsof the light chain variable region of SEQ ID NO:
 7. 5. The isolatedantibody or antigen binding fragment thereof of claim 1, wherein theisolated antibody comprises a heavy chain variable region consisting ofthe amino acids of the heavy chain variable region of SEQ ID NO: 9 and alight chain variable region consisting of the amino acids of the lightchain variable region of SEQ ID NO: 11.